Methods and compositions for the treatment of persistent infections and cancer by inhibiting the programmed cell death 1 (PD-1) pathway

ABSTRACT

The present invention provides methods and compositions for the treatment, prevention, or reduction of persistent infections, such as chronic infections, latent infections, and slow infections and cancer. The methods and compositions of the invention are also useful for the alleviation of one or more symptoms associated with such infections and cancer.

RELATED APPLICATIONS

This is a continuation of U.S. patent Ser. No. 15/252,133, filed on Aug.30, 2016, which is a continuation of U.S. patent application Ser. No.14/144,304, filed on Dec. 30, 2013, issued as U.S. Pat. No. 9,457,080,which is a continuation of U.S. patent application Ser. No. 11/449,919,filed on Jun. 8, 2006, issued as U.S. Pat. No. 8,652,465, which claimsthe benefit of U.S. Provisional Application No. 60/688,872, filed onJun. 8, 2005. The prior applications are incorporated herein byreference in their entirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under AI039671 andCA084500 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

FIELD OF THE INVENTION

In general, the present invention relates to methods and compositionsfor the treatment of persistent infections and cancer.

BACKGROUND OF THE INVENTION

Although the development of preventative vaccines has significantlyreduced the mortality rate of viral infections, the use of such vaccinesagainst viruses that cause persistent infections (e.g., hepatitis Cvirus) has been met with limited success. In contrast to viruses thatcause acute and self-limited infections, the immune response that ismounted against persistent infection-causing microbes is often transientand insufficient to clear the infection. As a result, the infectiousmicrobe remains within the infected subject for extended periods oftime, without necessarily causing constant host damage.

A major impediment in the eradication of persistent infection-causingmicrobes is the ability of such microbes to evade the immune system ofthe host organism. For example, certain viruses and parasitesdown-regulate the expression of host molecules necessary for efficient Tcell recognition of infected cells. Persistent infections also cause thefunctional impairment of antigen specific CD8+ T cells, which are vitalto the control and eradication of viral infections. Although thecombination of therapeutic vaccines with cytokine adjuvants has beenencouraging, the resulting immune responses have not successfullyeradicated the pathogen.

Thus, better methods are needed to treat, prevent, or alleviatepersistent infections.

SUMMARY OF THE INVENTION

The present invention provides methods and compositions for thetreatment, prevention, or reduction of, or alternatively the alleviationof one or more symptoms of, a persistent infection or cancer. Theinvention is based on the discovery that antigen specific CD8+ T cellsbecome functionally tolerant (‘exhausted’) to the infectious agentfollowing the induction of the Programmed Death-1 polypeptide (PD-1).Accordingly, by reducing the expression or activity of PD-1, PD-L1 orPDL2, the proliferation of functionally tolerant CD8+ T cells, theproduction of cytokines, and the rate of an infectious agent (e.g.,viral, bacterial, fungal, parasite, mycoplasm or cancer) clearance isincreased such that the immune response specific to the infectious agentis enhanced.

Accordingly, the invention provides a method of alleviating orpreventing a symptom of a persistent infection (e.g., a viral infection,a bacterial infection, a fungal infection, a mycoplasm infection and aparasitic infection) or cancer by administering to a subject in needthereof (e.g., a human) a compound that reduces the activity orexpression of a member of the CD28-like family (e.g., PD-1, CTLA-4, BTLAand a functional fragment or variant thereof) or CD28-like familyligands (e.g., PD-L1 or PD-L2). Alternatively, the subject isadministered an antigen-specific T cell or B cell that has beencontacted with an compound that reduces the expression or activity of aPD-1 polypeptide in the cell. For example, the antigen specific T cellor B cell is specific to a viral antigen. The T cell or B cell isderived from an autologous source or is derived from another subject ofthe same or different species as the subject being treated.

In addition, the invention features a method of increasing the cytotoxicactivity of a T cell (e.g., anergic T cell or T cell having increasedtolerance to antigens) by contacting the T cell with a compound thatreduces the activity or expression of a PD-1 polypeptide.

In all foregoing aspects of the invention, persistent viral infectionsresult from infections such as a hepatitis virus, a humanimmunodeficiency virus (HIV), a human T-lymphotrophic virus (HTLV), aherpes virus, an Epstein-Barr virus, or a human papilloma virus.Persistent viral infections may also include infections caused by alatent virus. Cancers include lymphoproliferative disorders such asangioimmunoblastic lymphoma and nodular lymphocyte Hodgkin lymphoma.Desirably, the compound of the invention increases an antigen specificimmune response by increasing the cytotoxic T-cell activity (e.g., anincrease in cytotoxic cytokine production such as IFNγ, TNFα, or IL-2,an increase in T cell proliferation, or an increase in viral clearance)in the subject being treated. For example, the compound reduces theexpression or activity of a PD ligand 1 (PD-L1) or a PD ligand 2 (PD-L2)or reduces the interaction between PD-1 and PD-L1 or the interactionbetween PD-1 and PD-L2. Exemplary compounds include antibodies (e.g., ananti-PD-1 antibody, an anti-PD-L1 antibody, and an anti-PD-L2 antibody),RNAi molecules (e.g., anti-PD-1 RNAi molecules, anti-PD-L1 RNAi, and ananti-PD-L2 RNAi), antisense molecules (e.g., an anti-PD-1 antisense RNA,an anti-PD-L1 antisense RNA, and an anti-PD-L2 antisense RNA), dominantnegative proteins (e.g., a dominant negative PD-1 protein, a dominantnegative PD-L1 protein, and a dominant negative PD-L2 protein), andsmall molecule inhibitors. Antibodies include monoclonal antibodies,humanized antibodies, deimmunized antibodies, and Ig fusion proteins. Anexemplary anti-PD-L1 antibody includes clone EH12.

In addition to the compound that reduces PD-1 expression or activity,the subject being treated may also be administered a vaccine that may ormay not include an adjuvant or a prime booster shot. Optionally, thesubject is administered a second compound, such as an antiviral compound(e.g., vidarabine, acyclovir, gancyclovir, valgancyclovir,nucleoside-analog reverse transcriptase inhibitor (NRTI) such as AZT(Zidovudine), ddI (Didanosine), ddC (Zalcitabine), d4T (Stavudine), or3TC (Lamivudine), non-nucleoside reverse transcriptase inhibitor (NNRTI)such as nevirapine or delavirdine, protease inhibitor such assaquinavir, ritonavir, indinavir, or nelfinavir, ribavirin, andinterferon), an antibacterial compound, an antifungal compound, anantiparasitic compound, an anti-inflammatory compound, anti-neoplasticcompounds or an analgesic. The second compound may also be a compoundthat reduces the expression or activity of cytotoxic T lymphocyteantigen 4 (CTLA-4) or B and T lymphocyte attenuator (BTLA). Otherexemplary compounds that may be administered to the subject areanti-CTLA-4 antibodies, anti-BTLA antibodies, anti-CD28 antibodies,anti-ICOS antibodies, anti-ICOS-L antibodies, anti-B7-1 antibodies,anti-B7-2 antibodies, anti-B7-H3 antibodies, and anti-B7-H4 antibodies.

The present invention further provides a method for identifying acandidate compound that modulates the activity or expression of a PD-1polypeptide that includes the steps of: (a) contacting a cell expressinga PD-1 gene (e.g., PD-1 fusion gene) with a candidate compound; (b)measuring the expression or activity of PD-1 in the cell (e.g., bymeasuring the expression of PD-1 mRNA or protein); and (c) comparing theexpression or activity of PD-1 in the cell compared to such expressionor activity in a control cell not contacted with the compound. Anincrease or decrease in the expression or activity of PD-1 indicates thecandidate compound as being useful for modulating the activity orexpression of a PD-1 polypeptide.

Alternatively, the screening method may involve the steps of: (a)contacting a T cell that overexpresses a PD-1 gene with a candidatecompound; and (b) determining the cytotoxic activity of the T cell; (c)comparing the cytotoxic activity of the T cell relative to such activityin a control cell not contacted with the compound. An increase ordecrease in such activity identifies the candidate compound as beinguseful for modulating the activity or expression of a PD-1 polypeptide.Cytotoxic activity includes cytokine production, T cell proliferation,and viral clearance.

The invention further provides a screening method involving the stepsof: (a) contacting a PD-1 polypeptide with a candidate compound; (b)determining whether the candidate compound interacts with the PD-1polypeptide; and (c) identifying a candidate compound as useful formodulating PD-1 expression or activity. Desirably, the identifiedcandidate compound interacts with the PD-1 polypeptide and reduces itsactivity.

The candidate compound identified by the screening methods describedherein may reduce the interaction between PD-1 and PD-L1 or theinteraction between PD-1 and PD-L2. The cell employed in any of thescreening methods described herein include mammalian cells such asrodent cells or human cell. The cell is an immune cell, such as a Tcell. Desirably, the PD-1 polypeptide used in such screening methods isa human PD-1 polypeptide.

Also provided herein is a method of diagnosing a subject as having or atrisk of having a persistent infection or cancer involving the steps of:(a) providing a sample containing immune cells (e.g., T cell or B cell)from a subject, and (b) measuring the expression or activity of PD-1 inthe sample. An increase in the expression or activity of PD-1 comparedto such expression or activity in a control sample identifies thesubject as having or at risk of having a persistent infection or cancer.Desirably, step (b) involves identifying antigen-specific immune cells,such as a viral antigen, bacterial antigen, parasitic antigen, or fungalantigen.

A method of selecting a treatment for a subject having or at risk ofhaving a persistent infection or cancer is also described. This methodinvolves the steps of: (a) providing a sample containing immune cells(e.g., T cell or B cell) from a subject; and (b) measuring theexpression or activity of PD-1 in the immune cells, such that anincrease in expression or activity of PD-1 compared to such expressionor activity in a control sample identifies the subject as having or atrisk of having a persistent infection or cancer; and (c) selecting atreatment for the subject diagnosed as having or at risk a persistentinfection or cancer, such that the treatment includes a compound thatreduces the expression or activity of PD-1. Desirably, step (b) involvesidentifying antigen-specific immune cells, such as a viral antigen,bacterial antigen, parasitic antigen, or fungal antigen.

Samples derived from subjects include blood samples, tissue biopsies,and bone marrow samples. Furthermore, control cells may be derived froma subject that does not have or at risk of having a persistentinfection.

The invention further provides a composition that contains: (a) acompound that reduces the level or activity of PD-1; and (b) a secondcompound, such as an antiviral compound, an antibacterial compound, anantifungal compound, an antiparasitic compound, an anti-inflammatorycompound, an analgesic, an anti-CTLA-4 antibody, an anti-BTLA antibody,an anti-CD28 antibody, an anti-ICOS antibody, an anti-ICOS-L antibody,an anti-B7-1 antibody, an anti-B7-2 antibody, an anti-B7-H3 antibody, oran anti-B7-H4 antibody.

The invention also provides a kit that contains (a) a compound thatreduces the level or activity of PD-1; and (b) instructions for deliveryof the compound to a subject. Alternatively, the kit contains (a) afirst compound that reduces the level or activity of PD-1; (b) a secondcompound such as an antiviral compound, an antibacterial compound, anantifungal compound, an antiparasitic compound, an anti-inflammatorycompound, an analgesic, an anti-CTLA-4 antibody, an anti-BTLA antibody,an anti-CD28 antibody, an anti-ICOS antibody, an anti-ICOS-L antibody,an anti-B7-1 antibody, an anti-B7-2 antibody, an anti-B7-H3 antibody, oran anti-B7-H4 antibody; and (c) instructions for delivery of the firstcompound and the second compound to a subject.

The present invention provides significant advantages over standardtherapies for treatment, prevention, and reduction, or alternatively,the alleviation of one or more symptoms of persistent infections.Administration of the therapeutic agent that reduces the level oractivity of PD-1 increases CD8+ T cell cytotoxicity, in turn increasingthe immune response to the infectious agent having the ability toestablish a persistent infection. In addition, the candidate compoundscreening methods provided by this invention allow for theidentification of novel therapeutics that modify the injury process,rather than merely mitigating the symptoms.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a bar graph showing the levels of PD-1 mRNA in D^(b)GP33-41and/or D^(b)GP276-286 specific T cells from naïve transgenic mice,lymphocytic choriomeningitis virus (LCMV) Armstrong immune(approximately 30 days post-infection) infected mice, or CD4-depletedLCMV-Cl-13 infected mice (approximately 30 days post-infection), asmeasured by gene array analysis.

FIG. 1B is a series of images of a flow cytometry experiment showingPD-1 surface expression on CD8+ tetramer+ T cells in LCMV Armstrongimmune and CD4 depleted LCMV-C1-13 infected mice approximately 60 dayspost-infection. Anergic CD8+ T cells express high levels of PD-1polypeptide on the cell surface approximately 60 days after chronicinfection with LCMV-Cl-13 virus (labeled “chronic”), but virus-specificCD8+ T cells do not express PD-1 polypeptide after clearance of an acuteLCMV Armstrong infection (labeled “immune”).

FIG. 1C is a series of images of a flow cytometry experimentdemonstrating the presence of PD-L1 on splenocytes from chronicallyinfected and uninfected mice. It demonstrates that PD-L1 expression isthe highest on the splenocytes that are infected by the virus.

FIG. 2A is a series of scatter plots showing that when C1-13 infectedmice are treated from day 23 to 37 post-infection there wasapproximately a 3 fold increase in the number of DbNP396-404 specificand DbGP33-41 specific CD8 T cells compared to the untreated controls.In order to determine any changes in function IFN-γ and TNF-α productionwas measured in response to 8 different LCMV epitopes.

FIG. 2B is a scatter plot showing that when all the known CD8 T cellspecificities are measured there is a 2.3 fold increase in total numberof LCMV specific CD8 T cells.

FIG. 2C is a series of flow cytometry graphs showing IFN-γ and TNF-αproduction in response to eight different LCMV epitopes.

FIG. 2D is a scatter plot showing that more virus specific CD8 T cellsin treated mice have the ability to produce TNF-α.

FIG. 2E is a series of bar charts showing that PD-L1 blockade alsoresulted in increased viral control in the spleen liver lung and serum.

FIG. 3A is a graph demonstrating the increase in DbGP33-41 andDbGP276-286 specific CD8+ T cells (labeled “GP33” and “GP276”) inCD4-depleted C1-13 infected mice treated with anti-PD-L1 (labeled“αPD-L”) from day 46 to day 60 post-infection versus control (labeled“untx”), which demonstrates that mice treated with anti-PD-L1 containedapproximately 7 fold more DbGP276-286 specific splenic CD8+ T cells andapproximately 4 fold more DbGP33-41 specific splenic CD8+ T cells thanuntreated mice.

FIG. 3B is a series of images demonstrating the increased frequency ofDbGP33-41 and DbGP276-286 specific CD8+ T cells in the spleen ofCD4-depleted C1-13 infected mice treated with anti-PD-L1 (labeled“αPD-L1 Tx”) from day 46 to day 60 post-infection versus control(labeled “untx”).

FIG. 3C is a series of images demonstrating increased proliferation ofDbGP276-286 specific CD8+ T cells in anti-PD-L-treated mice, as measuredby BrdU incorporation and Ki67 expression.

FIG. 3D is a chart showing that mice having high levels of CD8+ T cellexpansion demonstrate an appreciable response in peripheral bloodmononuclear cells (PBMC), as shown by comparing DbGP276-286 specificCD8+ T cells in the PBMC as compared to DbGP276-286 specific CD8+ Tcells in the spleen.

FIG. 4A is a series of charts demonstrating the increase in IFN-γproducing DbGP276-286 and DbGP33-41 specific CD8+ T cells inanti-PD-L-treated mice, as compared to controls. Higher frequencies ofDbNP396-404, KbNP205-212, DbNP166-175, and DbGP92-101 specific CD8+ Tcells were also detected in anti-PD-L-treated mice.

FIG. 4B is a chart demonstrating that in anti-PD-L-treated mice, 50% ofDbGP276-286 specific CD8+ T cells produce IFN-γ, as compared to 20% ofDbGP276-286 specific CD8+ T cells in control mice.

FIG. 4C is a series of images demonstrating that anti-PD-L-treatedchronically infected mice produce higher levels of TNF-α than untreatedchronically infected mice, but still produce lower levels of TNF-α thanimmune mice infected with LCMV Armstrong virus.

FIG. 4D is a chart demonstrating that treatment of LCMV-Cl-13 infectedmice with anti-PD-L1 renews ex vivo lytic activity of the virus-specificT cells, as compared to untreated infected mice, measured using a ⁵¹Crrelease assay.

FIG. 4E is a series of charts demonstrating the reduction of viraltiters in various organs following treatment of LCMV-Cl-13 infected micewith α-PD-L1. Viral titers decreased approximately 3 fold in the spleen,4 fold in the liver, 2 fold in the lung, and 2 fold in serum after 2weeks of anti-PD-L1 treatment, as compared to untreated mice.

FIG. 5A is a series of images of a flow cytometry experiment showingPD-1 surface expression using 10 HIV tetramers specific for dominantepitopes targeted in chronic clade C HIV infection. The percentagesindicate the percentage of tetramer⁺ cells that are PD-1⁺.

FIG. 5B is a series of charts demonstrating that the percentage and MFIof PD-1 is significantly upregulated on HIV-specific CD8 T cellscompared to the total CD8 T cell population (p<0.0001) in antiretroviraltherapy naïve individuals, and PD-1 is increased on the total CD8 T cellpopulation in HIV-infected versus HIV-seronegative controls (p=0.0033and p<0.0001, respectively). 120 HIV tetramer stains from 65HIV-infected individuals and 11 HIV seronegative controls were includedin the analysis.

FIG. 5C is a series of charts showing the median percentage and MFI ofPD-1 expression on tetramer⁺ cells by epitope specificity.

FIG. 5D is a chart depicting the variation in the percentage of PD-1⁺cells on different epitope-specific populations within individuals withmultiple detectable responses. Horizontal bars indicate the medianpercentage of PD-1⁺ HIV tetramer⁺ cells in each individual.

FIG. 6A is a series of charts demonstrating that there is no correlationbetween the number of HIV-specific CD8 T cells, as measured by tetramerstaining, and plasma viral load, whereas there is a positive correlationbetween both the percentage and MFI of PD-1 on tetramer⁺ cells andplasma viral load (p=0.0013 and p<0.0001, respectively).

FIG. 6B is a series of charts showing that there is no correlationbetween the number of HIV tetramer⁺ cells and CD4 count, whereas thereis an inverse correlation between the percentage and MFI of PD-1 on HIVtetramer⁺ cells and CD4 count (p=0.0046 and p=0.0150, respectively).

FIG. 6C is a series of charts demonstrating that the percentage and MFIof PD-1 on the total CD8 T cell population positively correlate withplasma viral load (p=0.0021 and p<0.0001, respectively).

FIG. 6D is a series of charts depicting the percentage and MFI of PD-1expression on the total CD8 T cell population is inversely correlatedwith CD4 count (p=0.0049 and p=0.0006, respectively).

FIG. 7A is a series of images of a flow cytometry experiment showingrepresentative phenotypic staining of B*4201 TL9-specific CD8 T cellsfrom subject SK222 in whom 98% of B*4201 TL9-specific CD8 T cells arePD-1⁺.

FIG. 7B is a chart illustrating a summary of phenotypic data frompersons in whom >95% of HIV-specific CD8 T cells are PD-1⁺. 7 to 19samples were analyzed for each of the indicated phenotypic markers. Thehorizontal bar indicates median percentage of tetramer⁺ PD-1⁺ cells thatwere positive for the indicated marker.

FIG. 8A is a series of images of a flow cytometry experiment showing therepresentative proliferation assay data from a B*4201 positive subject.After a 6-day stimulation with peptide, the percentage of B*4201TL9-specific CD8 T cells increased from 5.7% to 12.4% in the presence ofanti-PD-L1 blocking antibody.

FIG. 8B is a line graph depicting the summary proliferation assay dataindicating a significant increase in proliferation of HIV-specific CD8 Tcells in the presence of anti-PD-L1 blocking antibody (n=28, p=0.0006,paired t-test).

FIG. 8C is a bar graph showing the differential effects of PD-1/PD-L1blockade on proliferation of HIV-specific CD8 T cells on an individualpatient basis. White bars indicate fold increase of tetramer⁺ cells inthe presence of peptide alone, black bars indicate the fold increase oftetramer⁺ cells in the presence of peptide plus anti-PD-L1 blockingantibody. Individuals in whom CFSE assays were performed for more thanone epitope are indicated by asterisk, square, or triangle symbols.

SEQUENCE LISTING

The Sequence Listing is submitted as an ASCII text file6975-6975-99667-09_Sequence_Listing, Jun. 18, 2019, 2.72 KB], which isincorporated by reference herein.

DETAILED DESCRIPTION

The use of antibiotics and vaccines in recent decades has significantlyreduced the mortality rate due to microbial infections. The success ofantimicrobial treatment modalities, however, has been limited by theability of certain infectious agents to evade the immune system of thehost organism and in turn, establish a persistent infection. Forexample, the immune response that is mounted against viruses such ashepatitis and HIV is not sufficient to clear the infectious agent, whichremains in the infected subject. In such infections, antigen specificCD8+ T cells become functionally tolerant to the infectious agent, in astate known as ‘anergy’ or ‘exhaustion’. Anergic T cells lose theircytotoxic activity, i.e., their ability to produce cytokines,proliferate, and clear the infectious agent.

The present invention is based upon the surprising discovery that T cellanergy is concomitant with an induction in PD-1 expression and that PD-1expression correlates with certain types of lymphoproliferativedisorders. Accordingly, the invention provides methods of increasingT-cell cytoxicity by contacting a T-cell with an agent that reduces theexpression or activity of PD-1, PD-1 ligand (PD-L1) or PD-1 ligand 2(PD-L2). More specifically, the invention provides methods of treatingor preventing a persistent infection or lymphoproliferative disorders(e.g., cancers such as angioimmunoblastic lymphoma and nodularlymphocyte predominant Hodgkin lymphoma by administering to a subject anagent that reduces the expression or activity of PD-1. Reduction ofPD-1, PD-L1 or PD-L2 expression or activity results in an increase incytotoxic T cell activity, increasing the specific immune response tothe infectious agent. The results provided herein show that theadministration of anti-programmed death ligand-1 (PD-L1) blockingantibodies to persistently infected mice increased the cytotoxicactivity of anergic T cells. Specifically, disruption of PD-1 signalinginduced the expansion of anergic CD8+ T cells, enhanced cytokineproduction, and increased viral clearance. Furthermore, CD8+ T cellsgenerated during persistent infections of CD4 depleted mice proliferatedand regained much of their function upon anti-PD-L1 treatment.

In order for T cells to respond to foreign proteins, two signals must beprovided by antigen-presenting cells (APCs) to resting T lymphocytes.The first signal, which confers specificity to the immune response, istransduced via the T cell receptor (TCR) following recognition offoreign antigenic peptide presented in the context of the majorhistocompatibility complex (MHC). The second signal, termedcostimulation, induces T cells to proliferate and become functional.Costimulation is neither antigen-specific, nor MHC-restricted and isprovided by one or more distinct cell surface polypeptides expressed byAPCs. If T cells are only stimulated through the T cell receptor,without receiving an additional costimulatory signal, they becomenonresponsive, anergic, or die, resulting in downmodulation of theimmune response.

The CD80 (B7-1) and CD86 (B7-2) proteins, expressed on APCs, arecritical costimulatory polypeptides. While B7-2 plays a predominant roleduring primary immune responses, B7-1 is upregulated later in the courseof an immune response to prolong primary T cell responses orcostimulating secondary T cell responses. B7 polypeptides are capable ofproviding costimulatory or inhibitory signals to immune cells to promoteor inhibit immune cell responses. For example, when bound to acostimulatory receptor, PD-L1 (B7-4) induces costimulation of immunecells or inhibits immune cell costimulation when present in a solubleform. When bound to an inhibitory receptor, B7-4 molecules can transmitan inhibitory signal to an immune cell. Exemplary B7 family membersinclude B7-1, B7-2, B7-3 (recognized by the antibody BB-1), B7h (PD-L1),and B7-4 and soluble fragments or derivatives thereof. B7 family membersbind to one or more receptors on an immune cell, such as CTLA4, CD28,ICOS, PD-1 and/or other receptors, and, depending on the receptor, havethe ability to transmit an inhibitory signal or a costimulatory signalto an immune cell.

CD28 is a receptor that is constitutively expressed on resting T cells.After signaling through the T cell receptor, ligation of CD28 andtransduction of a costimulatory signal induces T cells to proliferateand secrete IL-2. CTLA4 (CD152), a receptor homologous to CD28, isabsent on resting T cells but its expression is induced following T cellactivation. CTLA4 plays a role in negative regulation of T cellresponses. ICOS, a polypeptide related to CD28 and CTLA4, is involved inIL-10 production. PD-1, the receptor to which PD-L1 and PD-L2 bind, isalso rapidly induced on the surface of T-cells. PD-1 is also expressedon the surface of B-cells (in response to anti-IgM) and on a subset ofthymocytes and myeloid cells.

Engagement of PD-1 (for example by crosslinking or by aggregation),leads to the transmission of an inhibitory signal in an immune cell,resulting in a reduction of immune responses concomitant with anincrease in immune cell anergy. PD-1 family members bind to one or morereceptors, such as PD-L1 and PD-L2 on antigen presenting cells.

PD-L1 and PD-L2, both of which are human PD-1 ligand polypeptides, aremembers of the B7 family of polypeptides. Each PD-1 ligand contains asignal sequence, an IgV domain, an IgC domain, a transmembrane domain,and a short cytoplasmic tail. These ligands are expressed in placenta,spleen, lymph nodes, thymus, and heart. PD-L2 is also expressed in thepancreas, lung, and liver, while PD-L1 is expressed in fetal liver,activated T-cells and endothelial cells. Both PD-1 ligands areupregulated on activated monocytes and dendritic cells.

Definitions

As used herein, by “persistent infection” is meant an infection in whichthe infectious agent (e.g., virus, bacterium, parasite, mycoplasm, orfungus) is not cleared or eliminated from the infected host, even afterthe induction of an immune response. Persistent infections may bechronic infections, latent infections, or slow infections. While acuteinfections are relatively brief (lasting a few days to a few weeks) andresolved from the body by the immune system, persistent infections maylast for months, years, or even a lifetime. These infections may alsorecur frequently over a long period of time, involving stages of silentand productive infection without cell killing or even producingexcessive damage to the host cells. The causative infectious agents mayalso be detected in the host (e.g., inside specific cells of infectedindividuals) even after the immune response has resolved, using standardtechniques. Mammals are diagnosed as having a persistent infectionaccording to any standard method known in the art and described, forexample, in U.S. Pat. Nos. 6,368,832, 6,579,854, and 6,808,710 and U.S.Patent Application Publication Nos. 20040137577, 20030232323,20030166531, 20030064380, 20030044768, 20030039653, 20020164600,20020160000, 20020110836, 20020107363, and 20020106730, all of which arehereby incorporated by reference. For example, a subject may bediagnosed as having a persistent Chlamydial infection following thedetection of Chlamydial species in a biological sample from thisindividual using PCR analysis. Mammals need not have not been diagnosedwith a persistent infection to be treated according to this invention.Microbial agents capable of establishing a persistent infection includeviruses (e.g., papilloma virus, hepatitis virus, human immune deficiencyvirus, and herpes virus), bacteria (e.g., Escherichia coli and Chlamydiaspp.), parasites, (e.g., Plasmodium, Leishmania spp., Schistosoma spp.,Trypanosoma spp., Toxoplasma spp.) and fungi.

By “alleviating a symptom of a persistent infection” is meantameliorating any of the conditions or symptoms associated with thepersistent infection before or after it has occurred. Alternatively,alleviating a symptom of a persistent infection may involve reducing theinfectious microbial (e.g., viral, bacterial, fungal, mycoplasm, orparasitic) load in the subject relative to such load in an untreatedcontrol. As compared with an equivalent untreated control, suchreduction or degree of prevention is at least 5%, 10%, 20%, 40%, 50%,60%, 80%, 90%, 95%, or 100% as measured by any standard technique.Desirably, the persistent infection is completely cleared as detected byany standard method known in the art, in which case the persistentinfection is considered to have been treated. A patient who is beingtreated for a persistent infection is one who a medical practitioner hasdiagnosed as having such a condition. Diagnosis may be by any suitablemeans. Diagnosis and monitoring may involve, for example, detecting thelevel of microbial load in a biological sample (e.g., tissue biopsy,blood test, or urine test), detecting the level of a surrogate marker ofthe microbial infection in a biological sample, detecting symptomsassociated with persistent infections, or detecting immune cellsinvolved in the immune response typical of persistent infections (e.g.,detection of antigen specific T cells that are anergic) A patient inwhom the development of a persistent infection is being prevented may ormay not have received such a diagnosis. One in the art will understandthat these patients may have been subjected to the same standard testsas described above or may have been identified, without examination, asone at high risk due to the presence of one or more risk factors (e.g.,family history or exposure to infectious agent).

As used herein, by “PD-1” is meant a polypeptide that forms a complexwith PD-L1 or PD-L2 proteins and is therefore involved in immuneresponses, such as the co-stimulation of T cells. The PD-1 proteins ofthe invention are substantially identical to the naturally occurringPD-1 (see, for example, Ishida et al. EMBO J. 11:3887-3895, 1992,Shinohara et al. Genomics 23:704-706, 1994; and U.S. Pat. No. 5,698,520,incorporated by reference herein). PD-1 signaling may reduce, forexample, CD8+ T cell cytoxicity by reducing T cell proliferation,cytokine production, or viral clearance. According to this invention,the PD-1 polypeptide reduces CD8+ T cell cytotoxic activity by at least5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more than 100% belowcontrol levels as measured by any standard method.

By a “PD-1 gene” is meant a nucleic acid that encodes a PD-1 protein.

By “PD-1 fusion gene” is meant a PD-1 promoter and/or all or part of aPD-1 coding region operably linked to a second, heterologous nucleicacid sequence. In preferred embodiments, the second, heterologousnucleic acid sequence is a reporter gene, that is, a gene whoseexpression may be assayed; reporter genes include, without limitation,those encoding glucuronidase (GUS), luciferase, chloramphenicoltransacetylase (CAT), green fluorescent protein (GFP), alkalinephosphatase, and .beta.-galactosidase.

By “reduce the expression or activity of PD-1” is meant to reduce thelevel or biological activity of PD-1 relative to the level or biologicalactivity of PD-1 in an untreated control. According to this invention,such level or activity is reduced by at least 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 100%, or even greater than 100%, relative to anuntreated control. For example, the biological activity of PD-1 isreduced if binding of PD-1 to PD-L1, PD-L2, or both is reduced, therebyresulting in a reduction in PD-1 signaling and therefore resulting in anincrease in CD8+ T cell cytotoxicity. As used herein, the term“activity” with respect to a PD-1 polypeptide includes any activitywhich is inherent to the naturally occurring PD-1 protein, such as theability to modulate an inhibitory signal in an activated immune cell,e.g., by engaging a natural ligand on an antigen presenting cell. Suchmodulation of an inhibitory signal in an immune cell results inmodulation of proliferation of and/or cytokine secretion by an immunecell. PD-1 may also modulate a costimulatory signal by competing with acostimulatory receptor for binding of a B7 molecule. Thus, the term“PD-1 activity” includes the ability of a PD-1 polypeptide to bind itsnatural ligand(s), the ability to modulate immune cell costimulatory orinhibitory signals, and the ability to modulate the immune response.Accordingly, reducing PD-1 activity includes reducing the interaction ofPD-1 to PD-L1 or PD-L2. This can be accomplished for example by blockingPD-L1 or PD-L2.

By “immune cell” is meant a cell of hematopoietic origin and that playsa role in the immune response. Immune cells include lymphocytes (e.g., Bcells and T cells), natural killer cells, and myeloid cells (e.g.,monocytes, macrophages, eosinophils, mast cells, basophils, andgranulocytes).

By “T cell” is meant a CD4+ T cell or a CD8+ T cell. The term T cellincludes both TH1 cells and TH2 cells.

The term “T cell cytoxicity” includes any immune response that ismediated by CD8+ T cell activation. Exemplary immune responses includecytokine production, CD8+ T cell proliferation, granzyme or perforinproduction, and clearance of the infectious agent.

By “unresponsiveness” includes refractivity of immune cells tostimulation, e.g., stimulation via an activating receptor or a cytokine.Unresponsiveness can occur, e.g., because of exposure toimmunosuppressants or exposure to high doses of antigen. As used herein,the term “anergy” or “tolerance” includes refractivity to activatingreceptor-mediated stimulation. Such refractivity is generallyantigen-specific and persists after exposure to the tolerizing antigenhas ceased. For example, anergy in T cells (as opposed tounresponsiveness) is characterized by lack of cytokine production, e.g.,IL-2. T cell anergy occurs when T cells are exposed to antigen andreceive a first signal (a T cell receptor or CD-3 mediated signal) inthe absence of a second signal (a costimulatory signal). Under theseconditions, re-exposure of the cells to the same antigen (even ifreexposure occurs in the presence of a costimulatory molecule) resultsin failure to produce cytokines and, thus, failure to proliferate.Anergic T cells can, however, mount responses to unrelated antigens andcan proliferate if cultured with cytokines (e.g., IL-2). For example, Tcell anergy can also be observed by the lack of IL-2 production by Tlymphocytes as measured by ELISA or by a proliferation assay using anindicator cell line. Alternatively, a reporter gene construct can beused. For example, anergic T cells fail to initiate IL-2 genetranscription induced by a heterologous promoter under the control ofthe 5′ IL-2 gene enhancer or by a multimer of the AP1 sequence that canbe found within the enhancer (Kang et al. Science 257:1134, 1992).Anergic antigen specific T cells may have a reduction of at least 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or even 100% in cytotoxicactivity relative a corresponding control antigen specific T cell.

By “purified antibody” is meant antibody which is at least 60%, byweight, free from proteins and naturally occurring organic moleculeswith which it is naturally associated. Preferably, the preparation is atleast 75%, more preferably 90%, and most preferably at least 99%, byweight, antibody, e.g., a PD-1, PD-L1, or PD-L2 specific antibody. Apurified antibody may be obtained, for example, by affinitychromatography using recombinantly-produced protein or conserved motifpeptides and standard techniques.

By “specifically binds” is meant an antibody that recognizes and bindsan antigen such as a PD-1, PD-L1, or PD-L2 polypeptide but that does notsubstantially recognize and bind other non-antigen molecules in asample, e.g., a biological sample, that naturally includes protein. Apreferred antibody binds to the PD-1, PD-L1, or PD-L2 polypeptidesdisclosed in U.S. Pat. No. 6,808,710 and U.S. Patent ApplicationPublication Nos. 20040137577, 20030232323, 20030166531, 20030064380,20030044768, 20030039653, 20020164600, 20020160000, 20020110836,20020107363, and 20020106730, all of which are hereby incorporated byreference.

By “neutralizing antibodies” is meant antibodies that interfere with anyof the biological activities of a PD-1 polypeptide, particularly theability of a PD-1 polypeptide to reduce an immune response such as thecytotoxicity of T cells. The neutralizing antibody may reduce theability of a PD-1 polypeptide to reduce an immune response by,preferably 50%, more preferably by 70%, and most preferably by 90% ormore. Any standard assay to measure immune responses, including thosedescribed herein, may be used to assess potentially neutralizingantibodies.

By “substantially identical,” when referring to a protein orpolypeptide, is meant a protein or polypeptide exhibiting at least 75%,but preferably 85%, more preferably 90%, most preferably 95%, or even99% identity to a reference amino acid sequence. For proteins orpolypeptides, the length of comparison sequences will generally be atleast 20 amino acids, preferably at least 30 amino acids, morepreferably at least 40 amino acids, and most preferably 50 amino acidsor the full length protein or polypeptide. Nucleic acids that encodesuch “substantially identical” proteins or polypeptides constitute anexample of “substantially identical” nucleic acids; it is recognizedthat the nucleic acids include any sequence, due to the degeneracy ofthe genetic code, that encodes those proteins or polypeptides. Inaddition, a “substantially identical” nucleic acid sequence alsoincludes a polynucleotide that hybridizes to a reference nucleic acidmolecule under high stringency conditions.

By “high stringency conditions” is meant any set of conditions that arecharacterized by high temperature and low ionic strength and allowhybridization comparable with those resulting from the use of a DNAprobe of at least 40 nucleotides in length, in a buffer containing 0.5 MNaHPO₄, pH 7.2, 7% SDS, 1 mM EDTA, and 1% BSA (Fraction V), at atemperature of 65° C., or a buffer containing 48% formamide, 4.8×SSC,0.2 M Tris-C1, pH 7.6, 1×Denhardt's solution, 10% dextran sulfate, and0.1% SDS, at a temperature of 42° C. Other conditions for highstringency hybridization, such as for PCR, Northern, Southern, or insitu hybridization, DNA sequencing, etc., are well known by thoseskilled in the art of molecular biology. See, e.g., F. Ausubel et al.,Current Protocols in Molecular Biology, John Wiley & Sons, New York,N.Y., 1998, hereby incorporated by reference.

By “substantially pure” is meant a nucleic acid, polypeptide, or othermolecule that has been separated from the components that naturallyaccompany it. Typically, the polypeptide is substantially pure when itis at least 60%, 70%, 80%, 90%, 95%, or even 99%, by weight, free fromthe proteins and naturally-occurring organic molecules with which it isnaturally associated. For example, a substantially pure polypeptide maybe obtained by extraction from a natural source, by expression of arecombinant nucleic acid in a cell that does not normally express thatprotein, or by chemical synthesis.

The term “isolated DNA” is meant DNA that is free of the genes which, inthe naturally occurring genome of the organism from which the given DNAis derived, flank the DNA. Thus, the term “isolated DNA” encompasses,for example, cDNA, cloned genomic DNA, and synthetic DNA.

By “an effective amount” is meant an amount of a compound, alone or in acombination, required to reduce or prevent hypertension or to treat orprevent a chronic infection in a mammal. The effective amount of activecompound(s) varies depending upon the route of administration, age, bodyweight, and general health of the subject. Ultimately, the attendingphysician or veterinarian will decide the appropriate amount and dosageregimen.

By a “candidate compound” is meant a chemical, be it naturally-occurringor artificially-derived. Candidate compounds may include, for example,peptides, polypeptides, synthetic organic molecules, naturally occurringorganic molecules, nucleic acid molecules, peptide nucleic acidmolecules, and components and derivatives thereof. For example, a usefulcandidate compound according to the present invention reduces binding ofPD-1 to PD-L1, PD-L2, or both.

The term “pharmaceutical composition” is meant any composition, whichcontains at least one therapeutically or biologically active agent andis suitable for administration to the patient. Any of these formulationscan be prepared by well-known and accepted methods of the art. See, forexample, Remington: The Science and Practice of Pharmacy, 20.sup.thedition, (ed. A R Gennaro), Mack Publishing Co., Easton, Pa., 2000.

Methods of Treating

T-cell cytotoxicity is increased by contacting a T-cell with a compoundthat reduces the expression or activity of PD-1. The T-cell is a naïveT-cell, memory T-cell or activated T-cell. Alternatively, the T-cell isan antigen specific T-cell. The antigen specific T cells is anergic ortolerant to the infectious agent. T-cell cytotoxicity is characterizedby an increase in cell proliferation and cytokine release.

The methods are useful to alleviate the symptoms of a variety ofinfections and cancers. An infection or cancer is treated, prevented ora symptom is alleviated by administering to a subject a PD-1 inhibitor.The subject is a mammal such as human, a primate, mouse, rat, dog, cat,cow, horse, and pig. The subject is suffering from or at risk ofdeveloping infection. A subject suffering from or at risk of developinginfection is by standard methods suitable for the particular infection.

The infection, e.g., bacterial, viral, fungal, mycoplasm, or parasiticis a persistent infection. Persistent infections, in contrast to acuteinfections are not effectively cleared by the induction of a host immuneresponse. The infectious agent and the immune response reach equilibriumsuch that the infected subject remains infectious over a long period oftime without necessarily expressing symptoms. Persistent infectionsinclude for example, latent, chronic and slow infections.

In a chronic infection, the infectious agent can be detected in the bodyat all times. However, the signs and symptoms of the disease may bepresent or absent for an extended period of time. Examples of chronicinfection include hepatitis B (caused by HBV) and hepatitis C (caused byHCV) adenovirus, cytomegalovirus, Epstein-Barr virus, herpes simplexvirus 1, herpes simplex virus 2, human herpesvirus 6, varicella-zostervirus, hepatitis B virus, hepatitis D virus, papilloma virus, parvovirusB19, polyomavirus BK, polyomavirus JC, measles virus, rubella virus,human immunodeficiency virus (HIV), human T cell leukemia virus I, andhuman T cell leukemia virus II. Parasitic persistent infections mayarise as a result of infection by Leishmania, Toxoplasma, Trypanosoma,Plasmodium, Schistosoma, and Encephalitozoon.

In a latent infection, the infectious agent (e.g., virus) is seeminglyinactive and dormant such that the subject does always exhibit signs orsymptoms. In a latent viral infection, the virus remains in equilibriumwith the host for long periods of time before symptoms again appear;however, the actual viruses cannot be detected until reactivation of thedisease occurs. Examples of latent infections include infections causedby HSV-1 (fever blisters), HSV-2 (genital herpes), and VZV(chickenpox-shingles).

In a slow infection, the infectious agents gradually increase in numberover a very long period of time during which no significant signs orsymptoms are observed. Examples of slow infections include AIDS (causedby HIV-1 and HIV-2), lentiviruses that cause tumors in animals, andprions.

In addition, persistent infections often arise as late complications ofacute infections. For example, subacute sclerosing panencephalitis(SSPE) can occur following an acute measles infection or regrossiveencephalitis can occur as a result of a rubella infection.

Cancers include for example angioimmunoblastic lymphoma or nodularlymphocyte predominant Hodgkin lymphoma.

Angioimmunoblastic lymphoma (AIL) is an aggressive (rapidly progressing)type of T-cell non-Hodgkin lymphoma marked by enlarged lymph nodes andhypergammaglobulinemia (increased antibodies in the blood). Othersymptoms may include a skin rash, fever, weight loss, positive Coomb'stest or night sweats. This malignancy usually occurs in adults. Patientsare usually aged 40-90 years (median around 65) and are more often male.As AIL progresses, hepatosplenomegaly, hemolytic anemia, and polyclonalhypergammaglobulinemia may develop. The skin is involved inapproximately 40-50% of patients.

Nodular lymphocyte predominant Hodgkin lymphoma is a B cell neoplasmthat appears to be derived from germinal center B cells with mutated,non-functional immunoglobulin genes. Similar to angioimmunoblasticlymphoma, neoplastic cells are associated with a meshwork of folliculardendritic cells. PD-1 expression is seen in T cells closely associatedwith neoplastic CD20+ cell in nodular lymphocyte predominant Hodgkinlymphoma, in a pattern similar to that seen for CD57+ T cells. CD57 hasbeen identified as another marker of germinal center-associated T cells,along with CXCR5, findings which support the conclusion that theneoplastic cells in nodular lymphocyte predominant Hodgkin lymphoma havea close association with germinal center-associated T cells.

An inhibitor of PD-1 is any agent having the ability to reduce theexpression or the activity of PD-1, PD-L1 or PD-2 in a cell. PD-1expression or activity is reduced by at least 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, or 100% compared to such expression or activity in acontrol cell. The control cell is a cell that has not been treated withthe PD-1 inhibitor. PD-1 expression or activity is determined by anystandard method in the art, including those described herein.Optionally, the PD-1 inhibitor inhibits or reduces binding of PD-1 toPD-L1, PD-L2, or both. PD-1 inhibitors include polypeptides,polynucleotides, small molecule antagonists, or siRNA.

A PD-1 inhibitor polypeptide includes, for example, an antibody orfragment thereof that reduces PD-1 expression or signaling. Exemplaryantibodies include anti-PD-1 antibodies, anti-PD-L1 antibodies,anti-PD-L2 antibodies, anti-CTLA-4 antibodies, anti-BTLA antibodies,anti-CD28 antibodies, anti-ICOS antibodies, anti-ICOS-L antibodies, ananti-B7-1 antibody, an anti-B7-2 antibody, anti-B7-H3 antibodies, oranti-B7-H4 antibodies.

Alternatively, the PD-1 inhibitor is a dominant negative protein or anucleic acid encoding a dominant negative protein that interferes withthe biological activity of PD-1 (i.e. binding of PD-1 to PD-L1, PD-L2,or both). A dominant negative protein is any amino acid molecule havinga sequence that has at least 50%, 70%, 80%, 90%, 95%, or even 99%sequence identity to at least 10, 20, 35, 50, 100, or more than 150amino acids of the wild type protein to which the dominant negativeprotein corresponds. For example, a dominant-negative PD-1 has mutationsuch that it no longer able to binds PD-L1.

The dominant negative protein may be administered as an expressionvector. The expression vector may be a non-viral vector or a viralvector (e.g., retrovirus, recombinant adeno-associated virus, or arecombinant adenoviral vector). Alternatively, the dominant negativeprotein may be directly administered as a recombinant proteinsystemically or to the infected area using, for example, microinjectiontechniques.

Small molecules includes, but are not limited to, peptides,peptidomimetics (e.g., peptoids), amino acids, amino acid analogs,polynucleotides, polynucleotide analogs, nucleotides, nucleotideanalogs, organic and inorganic compounds (including heterorganic andorganomettallic compounds) having a molecular weight less than about5,000 grams per mole, organic or inorganic compounds having a molecularweight less than about 2,000 grams per mole, organic or inorganiccompounds having a molecular weight less than about 1,000 grams permole, organic or inorganic compounds having a molecular weight less thanabout 500 grams per mole, and salts, esters, and other pharmaceuticallyacceptable forms of such compounds.

The PD-1 inhibitor is an antisense molecule, an RNA interference (siRNA)molecule, or a small molecule antagonist that targets PD-1 expression oractivity. By the term “siRNA” is meant a double stranded RNA moleculewhich prevents translation of a target mRNA. Standard techniques ofintroducing siRNA into a cell are used, including those in which DNA isa template from which an siRNA RNA is transcribed. The siRNA includes asense PD-1, PD-L1 or PD-L2 nucleic acid sequence, an anti-sense PD-1,PD-L1 or PD-L2 nucleic acid sequence or both. Optionally, the siRNA isconstructed such that a single transcript has both the sense andcomplementary antisense sequences from the target gene, e.g., a hairpin.Binding of the siRNA to an PD-1, PD-L1 or PD-L2 transcript in the targetcell results in a reduction in PD-1, PD-L1 or PD-L2 production by thecell. The length of the oligonucleotide is at least 10 nucleotides andmay be as long as the naturally-occurring PD-1, PD-L1 or PD-L2transcript. Preferably, the oligonucleotide is 19-25 nucleotides inlength. Most preferably, the oligonucleotide is less than 75, 50, 25nucleotides in length.

Other suitable PD-1 inhibitors are described in, for example, in U.S.Pat. No. 6,808,710 and U.S. Patent Application Publication Nos.20040137577, 20030232323, 20030166531, 20030064380, 20030044768,20030039653, 20020164600, 20020160000, 20020110836, 20020107363, and20020106730, all of which are hereby incorporated by reference.

The preferred dose of the PD-1 inhibitor is a biologically active dose.A biologically active dose is a dose that will induce an increase inCD8+ T cell cytotoxic activity the increase in the immune responsespecific to the infectious agent. Desirably, the PD-1 inhibitor has theability to reduce the expression or activity of PD-1 in antigen specificimmune cells (e.g., T cells such as CD8+ T cells) by at least 5%, 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more than 100% belowuntreated control levels. The levels or activity of PD-1 in immune cellsis measured by any method known in the art, including, for example,Western blot analysis, immunohistochemistry, ELISA, and Northern Blotanalysis. Alternatively, the biological activity of PD-1 is measured byassessing binding of PD-1 to PD-L1, PD-L2, or both. The biologicalactivity of PD-1 is determined according to its ability to increase CD8+T cell cytotoxicity including, for example, cytokine production,clearance of the infectious agent, and proliferation of antigen specificCD8+ T cells. Preferably, the agent that reduces the expression oractivity of PD-1 can increase the immune response specific to theinfectious agent by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, or more than 100% above untreated control levels. The agent of thepresent invention is therefore any agent having any one or more of theseactivities. Although the agent of the invention is preferably expressedin CD8+ T cells, it is understood that any cell that can influence theimmune response to persistent infections is also amenable to the methodsof the invention and include, for example, B cells.

Optionally, the subject is administered one or more additionaltherapeutic agents. Additional therapeutic agents include, for example,antiviral compounds (e.g., vidarabine, acyclovir, gancyclovir,valgancyclovir, nucleoside-analog reverse transcriptase inhibitor (NRTI)(e.g., AZT (Zidovudine), ddI (Didanosine), ddC (Zalcitabine), d4T(Stavudine), or 3TC (Lamivudine)), non-nucleoside reverse transcriptaseinhibitor (NNRTI) (e.g., (nevirapine or delavirdine), protease inhibitor(saquinavir, ritonavir, indinavir, or nelfinavir), ribavirin, orinterferon), antibacterial compounds, antifungal compounds,antiparasitic compounds, anti-inflammatory compounds, anti-neoplasticagent or analgesics.

The additional therapeutic agent is administered prior to,concomitantly, or subsequent to administration of the PD-1 inhibitor.For example, the PD-1 inhibitor and the additional agent areadministered in separate formulations within at least 1, 2, 4, 6, 10,12, 18, or more than 24 hours apart. Optionally, the additional agent isformulated together with the PD-1 inhibitor. When the additional agentis present in a different composition, different routes ofadministration may be used. The agent is administered at doses known tobe effective for such agent for treating, reducing, or preventing aninfection.

Concentrations of the PD-1 inhibitor and the additional agent dependsupon different factors, including means of administration, target site,physiological state of the mammal, and other medication administered.Thus treatment dosages may be titrated to optimize safety and efficacyand is within the skill of an artisan. Determination of the properdosage and administration regime for a particular situation is withinthe skill of the art.

Optionally, the subject is further administered a vaccine that elicits aprotective immune response against the infectious agent that causes apersistent infection. For example, the subject receives a vaccine thatelicits an immune response against human immunodeficiency virus (HIV),tuberculosis, influenza, or hepatitis C. Exemplary vaccines aredescribed, for example, in Berzofsky et al. (J. Clin. Invest.114:456-462, 2004). If desired, the vaccine is administered with aprime-booster shot or with adjuvants.

PD-1 inhibitors are administered in an amount sufficient to increase Tcell, e.g., CD8+ T cell, cytotoxicity. An increase in T-cellcytotoxicity results in an increased immune response and a reduction inthe persistent infection. An increased immune response is measured, forexample, by an increase in immune cell proliferation, e.g., T-cell or Bcell, an increase in cytokine production, and an increase in theclearance of an infectious agent. Such reduction includes thealleviation of one or more of symptoms associated with the persistentinfection. Administration of the PD-1 inhibitor reduces the persistentinfection or alleviates one or more symptoms associated with thepersistent infection by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, or 100% as compared to an untreated subject.

Treatment is efficacious if the treatment leads to clinical benefit suchas, a reduction of the load of the infectious agent in the subject. Whentreatment is applied prophylactically, “efficacious” means that thetreatment retards or prevents an infection from forming. Efficacy may bedetermined using any known method for diagnosing or treating theparticular infection.

Therapeutic Administration

The invention includes administering to a subject a composition thatincludes a compound that reduces PD-1 expression or activity (referredto herein as an “PD-1 inhibitor” or “therapeutic compound”).

An effective amount of a therapeutic compound is preferably from about0.1 mg/kg to about 150 mg/kg. Effective doses vary, as recognized bythose skilled in the art, depending on route of administration,excipient usage, and coadministration with other therapeutic treatmentsincluding use of other anti-infection agents or therapeutic agents fortreating, preventing or alleviating a symptom of a particular infectionor cancer. A therapeutic regimen is carried out by identifying a mammal,e.g., a human patient suffering from (or at risk of developing) aninfection or cancer, using standard methods.

The pharmaceutical compound is administered to such an individual usingmethods known in the art. Preferably, the compound is administeredorally, rectally, nasally, topically or parenterally, e.g.,subcutaneously, intraperitoneally, intramuscularly, and intravenously.The compound is administered prophylactically, or after the detection ofan infection. The compound is optionally formulated as a component of acocktail of therapeutic drugs to treat infection. Examples offormulations suitable for parenteral administration include aqueoussolutions of the active agent in an isotonic saline solution, a 5%glucose solution, or another standard pharmaceutically acceptableexcipient. Standard solubilizing agents such as PVP or cyclodextrins arealso utilized as pharmaceutical excipients for delivery of thetherapeutic compounds.

The therapeutic compounds described herein are formulated intocompositions for other routes of administration utilizing conventionalmethods. For example, PD-1 inhibitor is formulated in a capsule or atablet for oral administration. Capsules may contain any standardpharmaceutically acceptable materials such as gelatin or cellulose.Tablets may be formulated in accordance with conventional procedures bycompressing mixtures of a therapeutic compound with a solid carrier anda lubricant. Examples of solid carriers include starch and sugarbentonite. The compound is administered in the form of a hard shelltablet or a capsule containing a binder, e.g., lactose or mannitol, aconventional filler, and a tableting agent. Other formulations includean ointment, suppository, paste, spray, patch, cream, gel, resorbablesponge, or foam. Such formulations are produced using methods well knownin the art.

Where the therapeutic compound is a nucleic acid encoding a protein, theTherapeutic nucleic acid is administered in vivo to promote expressionof its encoded protein, by constructing it as part of an appropriatenucleic acid expression vector and administering it so that it becomesintracellular (e.g., by use of a retroviral vector, by direct injection,by use of microparticle bombardment, by coating with lipids orcell-surface receptors or transfecting agents, or by administering it inlinkage to a homeobox-like peptide which is known to enter the nucleus(See, e.g., Joliot, et al., 1991. Proc Natl Acad Sci USA 88:1864-1868),and the like. Alternatively, a nucleic acid therapeutic is introducedintracellularly and incorporated within host cell DNA for expression, byhomologous recombination or remain episomal.

For local administration of DNA, standard gene therapy vectors used.Such vectors include viral vectors, including those derived fromreplication-defective hepatitis viruses (e.g., HBV and HCV),retroviruses (see, e.g., WO 89/07136; Rosenberg et al., 1990, N. Eng. J.Med. 323(9):570-578), adenovirus (see, e.g., Morsey et al., 1993, J.Cell. Biochem., Supp. 17E,), adeno-associated virus (Kotin et al., 1990,Proc. Natl. Acad. Sci. USA 87:2211-2215,), replication defective herpessimplex viruses (HSV; Lu et al., 1992, Abstract, page 66, Abstracts ofthe Meeting on Gene Therapy, September 22-26, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.), and any modified versions ofthese vectors. The invention may utilize any other delivery system whichaccomplishes in vivo transfer of nucleic acids into eucaryotic cells.For example, the nucleic acids may be packaged into liposomes, e.g.,cationic liposomes (Lipofectin), receptor-mediated delivery systems,non-viral nucleic acid-based vectors, erytbrocyte ghosts, ormicrospheres (e.g., microparticles; see, e.g., U.S. Pat. Nos. 4,789,734;4,925,673; 3,625,214; Gregoriadis, 1979, Drug Carriers in Biology andMedicine, pp. 287-341 (Academic Press,). Naked DNA may also beadministered.

DNA for gene therapy can be administered to patients parenterally, e.g.,intravenously, subcutaneously, intramuscularly, and intraperitoneally.DNA or an inducing agent is administered in a pharmaceuticallyacceptable carrier, i.e., a biologically compatible vehicle which issuitable for administration to an animal e.g., physiological saline. Atherapeutically effective amount is an amount which is capable ofproducing a medically desirable result, e.g., an decrease of a PD-1 geneproduct in a treated animal. Such an amount can be determined by one ofordinary skill in the art. As is well known in the medical arts, dosagefor any given patient depends upon many factors, including the patient'ssize, body surface area, age, the particular compound to beadministered, sex, time and route of administration, general health, andother drugs being administered concurrently. Dosages may vary, but apreferred dosage for intravenous administration of DNA is approximately10⁶ to 10²² copies of the DNA molecule. Typically, plasmids areadministered to a mammal in an amount of about 1 nanogram to about 5000micrograms of DNA. Desirably, compositions contain about 5 nanograms to1000 micrograms of DNA, 10 nanograms to 800 micrograms of DNA, 0.1micrograms to 500 micrograms of DNA, 1 microgram to 350 micrograms ofDNA, 25 micrograms to 250 micrograms of DNA, or 100 micrograms to 200micrograms of DNA. Alternatively, administration of recombinantadenoviral vectors encoding the PD-1 inhibitor into a mammal may beadministered at a concentration of at least 10⁵, 10⁶, 10⁷, 10⁸, 10⁹,10¹⁰, or 10¹¹ plaque forming unit (pfu).

PD-1 gene products are administered to the patient intravenously in apharmaceutically acceptable carrier such as physiological saline.Standard methods for intracellular delivery of peptides can be used,e.g. packaged in liposomes. Such methods are well known to those ofordinary skill in the art. It is expected that an intravenous dosage ofapproximately 1 to 100 moles of the polypeptide of the invention wouldbe administered per kg of body weight per day. The compositions of theinvention are useful for parenteral administration, such as intravenous,subcutaneous, intramuscular, and intraperitoneal.

PD-1 inhibitors are effective upon direct contact of the compound withthe affected tissue. Accordingly, the compound may be administeredtopically. Alternatively, PD-1 inhibitors may be administeredsystemically. Additionally, compounds may be administered by implanting(either directly into an organ (e.g., intestine or liver) orsubcutaneously) a solid or resorbable matrix which slowly releases thecompound into adjacent and surrounding tissues of the subject. Forexample, for the treatment of gastrointestinal infection, the compoundmay be administered systemically (e.g., intravenously, rectally ororally) or locally (e.g., directly into gastric tissue). Alternatively,a PD-1 inhibitor-impregnated wafer or resorbable sponge is placed indirect contact with gastric tissue. The PD-1 inhibitor is slowlyreleased in vivo by diffusion of the drug from the wafer and erosion ofthe polymer matrix. As another example, infection of the liver (i.e.,hepatitis) is treated by infusing into the liver vasculature a solutioncontaining the PD-1 inhibitor.

For the treatment of neurological infections, the PD-1 inhibitor may beadministered intravenously or intrathecally (i.e., by direct infusioninto the cerebrospinal fluid). For local administration, acompound-impregnated wafer or resorbable sponge is placed in directcontact with CNS tissue. The compound or mixture of compounds is slowlyreleased in vivo by diffusion of the drug from the wafer and erosion ofthe polymer matrix. Alternatively, the compound is infused into thebrain or cerebrospinal fluid using standard methods. For example, a burrhole ring with a catheter for use as an injection port is positioned toengage the skull at a burr hole drilled into the skull. A fluidreservoir connected to the catheter is accessed by a needle or styletinserted through a septum positioned over the top of the burr hole ring.A catheter assembly (described, for example, in U.S. Pat. No. 5,954,687)provides a fluid flow path suitable for the transfer of fluids to orfrom selected location at, near or within the brain to allowadministration of the drug over a period of time.

For cardiac infections, the compound may be delivered, for example, tothe cardiac tissue (i.e., myocardium, pericardium, or endocardium) bydirect intracoronary injection through the chest wall or using standardpercutaneous catheter based methods under fluoroscopic guidance. Thus,the inhibitor may be directly injected into tissue or may be infusedfrom a stent or catheter which is inserted into a bodily lumen. Anyvariety of coronary catheter or perfusion catheter may be used toadminister the compound. Alternatively, the compound is coated orimpregnated on a stent that is placed in a coronary vessel.

Pulmonary infections may be treated, for example, by administering thecompound by inhalation. The compounds are delivered in the form of anaerosol spray from a pressured container or dispenser which contains asuitable propellant, e.g., a gas such as carbon dioxide or a nebulizer.

One in the art will understand that the patients treated according tothe invention may have been subjected to the same tests to diagnose apersistently infected subject or may have been identified, withoutexamination, as one at high risk due to the presence of one or more riskfactors (e.g., exposure to infectious agent, exposure to infectedsubject, genetic predisposition, or having a pathological conditionpredisposing to secondary infections). Reduction of persistent infectionsymptoms or damage may also include, but are not limited to, alleviationof symptoms, diminishment of extent of disease, stabilized (i.e., notworsening) state of disease, delay or slowing of disease progression,and amelioration or palliation of the disease state. Treatment may occurat home with close supervision by the health care provider, or may occurin a health care facility.

Methods to Measure Immune Response

Methods for measuring the immune response following treatment accordingto the present invention are well known in the art. The activity of Tcells may be assessed, for example, by assays that detect cytokineproduction, assays measuring T cell proliferation, assays that measurethe clearance of the microbial agent, and assays that measure CD8+ Tcell cytotoxicity. These assays are described, for example, in U.S. Pat.No. 6,808,710 and U.S. Patent Application Publication Nos. 20040137577,20030232323, 20030166531, 20030064380, 20030044768, 20030039653,20020164600, 20020160000, 20020110836, 20020107363, and 20020106730, allof which are hereby incorporated by reference.

Optionally, the ability of a PD-1 inhibitor to increase CD8+ T cellcytotoxicity is assessed by assays that measure the proliferation ofCD8+ T cells (e.g., thymidine incorporation, BrdU assays, and stainingwith cell cycle markers (e.g., Ki67 and CFSE), described, for example,by Dong et al (Nature 5:1365-1369, 1999). In one example, T-cellproliferation is monitored by culturing the purified T-cells expressingPD-1 with a PD-1 inhibitor, a primary activation signal as describedabove, and ³H-thymidine. The level of T-cell proliferation is determinedby measuring thymidine incorporation.

CD8+ T cell cytotoxicity is also assessed by lysis assays (e.g., 51Crrelease assays or assays detecting the release of perforin or granzyme),assays that detect caspase activation, or assays that measure theclearance of the microbial agent from the infected subject. For example,the viral load in a biological sample from the infected subject (e.g.,serum, spleen, liver, lung, or the tissue to which the virus is tropic)may be measured before and after treatment.

The production of cytokines such as IFNγ, TNF-α, and IL-2 may also bemeasured. For example, purified T-cells are cultured in the presence ofthe PD-1 inhibitor protein and a primary activation signal. The level ofvarious cytokines in the supernatant can be determined by sandwichenzyme-linked immunosorbent assays or other conventional assaysdescribed, for example, in Dong et al (Nature 5:1365-1369, 1999).

If desired, the efficacy of the PD-1 inhibitor is assessed by itsability to induce co-stimulation of T cells. For example, a method forin vitro T-cell co-stimulation involves providing purified T-cells thatexpress PD-1 with a first or primary activation signal by anti-CD3monoclonal antibody or phorbol ester, or by antigen in association withclass II MHC. The ability of a candidate compound agent to reduce PD-1expression or activity and therefore provide the secondary orco-stimulatory signal necessary to modulate immune function, to theseT-cells can then be assayed by any one of the several conventionalassays well known in the art.

A B cell response is assessed by an antigen specific ELISA (e.g., LCMV,HIV, tuberculosis, or malaria), plasma cell ELISPOT, memory B-cellassay, phenotyping of B cell, and analysis of germinal centers byimmunohistochemistry.

Screening Assays

The present invention provides screening methods to identify compoundsthat can inhibit the expression or activity of PD-1. Useful compoundsinclude any agent that inhibits the biological activity or reduces thecellular level of PD-1. For example, candidate compounds may reducebinding of PD-1 to PD-L1, PD-L2, or both. Using such agents as leadcompounds, for example, the present screening methods also allow theidentification of further novel, specific inhibitors of PD-1 thatfunction to treat, reduce, or prevent persistent infections, oralternatively, that alleviate one or more symptoms associated with suchinfections. The method of screening may involve high-throughputtechniques.

By a “candidate compound” is meant a chemical, be it naturally-occurringor artificially-derived. Candidate compounds may include, for example,peptides, polypeptides, synthetic organic molecules, naturally occurringorganic molecules, nucleic acid molecules, peptide nucleic acidmolecules, and components and derivatives thereof. For example, a usefulcandidate compound according to the present invention reduces binding ofPD-1 to PD-L1, PD-L2, or both.

A number of methods are available for carrying out such screeningassays. According to one approach, candidate compounds are added atvarying concentrations to the culture medium of cells expressing PD-1.By a “PD-1 gene” is meant a nucleic acid that encodes a PD-1 protein. By“PD-1 fusion gene” is meant a PD-1 promoter and/or all or part of a PD-1coding region operably linked to a second, heterologous nucleic acidsequence. In preferred embodiments, the second, heterologous nucleicacid sequence is a reporter gene, that is, a gene whose expression maybe assayed; reporter genes include, without limitation, those encodingglucuronidase (GUS), luciferase, chloramphenicol transacetylase (CAT),green fluorescent protein (GFP), alkaline phosphatase, andbeta-galactosidase.

Gene expression of PD-1 is then measured, for example, by standardNorthern blot analysis (Ausubel et al., supra), using any appropriatefragment prepared from the nucleic acid molecule of PD-1 as ahybridization probe or by real time PCR with appropriate primers. Thelevel of gene expression in the presence of the candidate compound iscompared to the level measured in a control culture medium lacking thecandidate molecule. If desired, the effect of candidate compounds may,in the alternative, be measured at the level of PD-1 polypeptide usingthe same general approach and standard immunological techniques, such asWestern blotting or immunoprecipitation with an antibody specific toPD-1 for example. For example, immunoassays may be used to detect ormonitor the level of PD-1. Polyclonal or monoclonal antibodies which arecapable of binding to PD-1 may be used in any standard immunoassayformat (e.g., ELISA or RIA assay) to measure the levels of PD-1. PD-1can also be measured using mass spectroscopy, high performance liquidchromatography, spectrophotometric or fluorometric techniques, orcombinations thereof.

Alternatively, the screening methods of the invention may be used toidentify candidate compounds that decrease the biological activity ofPD-1 by reducing binding of PD-1 to PD-L1, PD-L2, or both by at least10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% relative to anuntreated control. For example, a candidate compound may be tested forits ability to decrease PD-1 activity in cells that naturally expressPD-1, after transfection with cDNA for PD-1, or in cell-free solutionscontaining PD-1, as described further below. The effect of a candidatecompound on the binding or activation of PD-1 can be tested byradioactive and non-radiaoctive binding assays, competition assays, andreceptor signaling assays.

As a specific example, mammalian cells (e.g., rodent cells) that expressa nucleic acid encoding PD-1 are cultured in the presence of a candidatecompound (e.g., a peptide, polypeptide, synthetic organic molecule,naturally occurring organic molecule, nucleic acid molecule, orcomponent thereof). Cells may either endogenously express PD-1 or mayalternatively be genetically engineered by any standard technique knownin the art (e.g., transfection and viral infection) to overexpress PD-1.The expression level of PD-1 is measured in these cells by means ofWestern blot analysis and subsequently compared to the level ofexpression of the same protein in control cells that have not beencontacted by the candidate compound. A compound which promotes adecrease in the level of PD-1 activity as a result of reducing itssynthesis or biological activity is considered useful in the invention.

In one particular example, a compound that interferes with PD-1 bindingto PD-L, PD-L2, or both (thereby reducing the biological activity ofPD-1), leading to an increase in an immune response, is useful accordingto the present invention. Given its ability to decrease the biologicalactivity of PD-1, such a molecule may be used, for example, as atherapeutic agent to treat, reduce, or prevent a persistent infection,or alternatively, to alleviate one or more symptoms associated with suchinfections. As a specific example, a candidate compound may be contactedwith two proteins, the first protein being a polypeptide substantiallyidentical to PD-1 and the second protein being either PD-L1 or PD-L2(i.e., a protein that binds the PD-1 polypeptide under conditions thatallow binding and that results in a reduced immune response). Accordingto this particular screening method, the interaction between these twoproteins is measured following the addition of a candidate compound. Adecrease in the binding PD-1 to the second polypeptide following theaddition of the candidate compound (relative to such binding in theabsence of the compound) identifies the candidate compound as having theability to inhibit the interaction between the two proteins. Ultimately,the screening assay of the invention may be carried out, for example, ina cell-free system or using a yeast two-hybrid system. If desired, oneof the proteins or the candidate compound may be immobilized on asupport as described above or may have a detectable group.

Alternatively, or in addition, candidate compounds may be screened forthose which specifically bind to and thereby inhibit PD-1. The efficacyof such a candidate compound is dependent upon its ability to interactwith PD-1. Such an interaction can be readily assayed using any numberof standard binding techniques and functional assays (e.g., thosedescribed in Ausubel et al., supra). For example, a candidate compoundmay be tested in vitro for interaction and binding with PD-1 and itsability to modulate immune responses may be assayed by any standardassays (e.g., those described herein).

For example, a candidate compound that binds to PD-1 may be identifiedusing a chromatography-based technique. For example, a recombinant PD-1may be purified by standard techniques from cells engineered to expressPD-1 (e.g., those described above) and may be immobilized on a column.Alternatively, the naturally-occurring PD-1 may be immobilized on acolumn. A solution of candidate compounds is then passed through thecolumn, and a compound specific for PD-1 is identified on the basis ofits ability to bind to PD-1 and be immobilized on the column. To isolatethe compound, the column is washed to remove non-specifically boundmolecules, and the compound of interest is then released from the columnand collected. Compounds isolated by this method (or any otherappropriate method) may, if desired, be further purified (e.g., by highperformance liquid chromatography).

Screening for new inhibitors and optimization of lead compounds may beassessed, for example, by assessing their ability to modulate cytotoxicT cell activity or the immune response using standard techniques. Inaddition, these candidate compounds may be tested for their ability tofunction as anti-microbial agents (e.g., as described herein). Compoundsisolated by this approach may also be used, for example, as therapeuticsto treat, reduce, or prevent persistent infections, or alternatively, toalleviate one or more symptoms associated with such infections.Compounds which are identified as binding to PD-1 with an affinityconstant less than or equal to 10 mM are considered particularly usefulin the invention.

Potential therapeutic agents include organic molecules, peptides,peptide mimetics, polypeptides, and antibodies that bind to a nucleicacid sequence or polypeptide that encodes PD-1 and thereby inhibit orextinguish their activity. Potential anti-microbial agents also includesmall molecules that bind to and occupy the binding site of suchpolypeptides thereby preventing binding to cellular binding molecules,such that normal biological activity is prevented. Other potentialanti-microbial agents include antisense molecules.

Diagnostic and Prognostic Methods

Cancer, e.g. angioimmunoblastic T cell lymphoma or Nodular LymphocytePredominant Hodgkin's Lymphoma are detected by examining the amount of aPD-1 polypeptide in a test sample (i.e., a patient derived sample). Achange in the level if the PD-1 polypeptide compared to a control sampleis indicative of cancer in the subject. The change may be an increase ora decrease in the PD-1 polypeptide relative to a control sample. Thecontrol sample is prepared (i.e., fractionated) in a similar fashion asthe test sample.

A sample is for example, blood, serum, acsites fluid, urine, or otherbodily fluids. Preferably the sample is a T-cell or a B-cell

The amount of the PD-1 is determined in the test sample and compared tothe expression of the normal control level. By normal control level ismeant the expression level of a PD-1 polypeptide typically found in asubject not suffering from a cancer. An increase of the level in thepatient derived sample of a PD-1 indicates that the subject is sufferingfrom or is at risk of developing cancer. In contrast, when the methodsare applied prophylacticly, a similar level or a decrease in the levelin the patient derived sample of a PD-1 polypeptide indicates that thesubject is not suffering from or is at risk of developing cancer. Anincrease of the level in the patient derived sample of a PD-1polypeptide indicates that the subject is suffering from or is at riskof developing cancer.

The alteration in the amount of the PD-1 polypeptide is statisticallysignificant. By statistically significant is meant that the alterationis greater than what might be expected to happen by change alone.Statistical significance is determined by method known in the art. Forexample statistical significance is determined by p-value. The p-valuesis a measure of probability that a difference between groups during anexperiment happened by chance. (P(z≥z_(observed))). For example, ap-value of 0.01 means that there is a 1 in 100 chance the resultoccurred by chance. The lower the p-value, the more likely it is thatthe difference between groups was caused by treatment. An alteration isstatistically significant if the p-value is at least 0.05. Preferably,the p-value is 0.04, 0.03, 0.02, 0.01, 0.005, 0.001 or less.

The “diagnostic accuracy” of a test, assay, or method concerns theability of the test, assay, or method to distinguish between patientshaving cancer or at risk for cancer is based on whether the patientshave a “clinically significant presence” of a PD-1 polypeptide. By“clinically significant presence” is meant that the presence of the PD-1polypeptide in the patient (typically in a sample from the patient) ishigher or lower than the predetermined cut-off point (or thresholdvalue) for that PD-1 polypeptide and therefore indicates that thepatient has cancer for which the sufficiently high presence of thatprotein is a marker.

The terms “high degree of diagnostic accuracy” and “very high degree ofdiagnostic accuracy” refer to the test or assay for that PD-1polypeptide with the predetermined cut-off point correctly (accurately)indicating the presence or absence of the cancer. A perfect test wouldhave perfect accuracy. Thus, for individuals who have diabetes, the testwould indicate only positive test results and would not report any ofthose individuals as being “negative” (there would be no “falsenegatives”). In other words, the “sensitivity” of the test (the truepositive rate) would be 100%. On the other hand, for individuals who didnot have diabetes, the test would indicate only negative test resultsand would not report any of those individuals as being “positive” (therewould be no “false positives”). In other words, the “specificity” (thetrue negative rate) would be 100%. See, e.g., O'Marcaigh A S, Jacobson RM, “Estimating The Predictive Value Of A Diagnostic Test, How To PreventMisleading Or Confusing Results,” Clin. Ped. 1993, 32(8): 485-491, whichdiscusses specificity, sensitivity, and positive and negative predictivevalues of a test, e.g., a clinical diagnostic test.

Changing the cut point or threshold value of a test (or assay) usuallychanges the sensitivity and specificity but in a qualitatively inverserelationship. For example, if the cut point is lowered, more individualsin the population tested will typically have test results over the cutpoint or threshold value. Because individuals who have test resultsabove the cut point are reported as having the disease, condition, orsyndrome for which the test is being run, lowering the cut point willcause more individuals to be reported as having positive results (i.e.,that they have cancer). Thus, a higher proportion of those who havecancer will be indicated by the test to have it. Accordingly, thesensitivity (true positive rate) of the test will be increased. However,at the same time, there will be more false positives because more peoplewho do not have the disease, condition, or syndrome (i.e., people whoare truly “negative”) will be indicated by the test to have PD-1polypeptide values above the cut point and therefore to be reported aspositive (i.e., to have the disease, condition, or syndrome) rather thanbeing correctly indicated by the test to be negative. Accordingly, thespecificity (true negative rate) of the test will be decreased.Similarly, raising the cut point will tend to decrease the sensitivityand increase the specificity. Therefore, in assessing the accuracy andusefulness of a proposed medical test, assay, or method for assessing apatient's condition, one should always take both sensitivity andspecificity into account and be mindful of what the cut point is atwhich the sensitivity and specificity are being reported becausesensitivity and specificity may vary significantly over the range of cutpoints.

There is, however, an indicator that allows representation of thesensitivity and specificity of a test, assay, or method over the entirerange of cut points with just a single value. That indicator is derivedfrom a Receiver Operating Characteristics (“ROC”) curve for the test,assay, or method in question. See, e.g., Shultz, “ClinicalInterpretation Of Laboratory Procedures,” chapter 14 in Teitz,Fundamentals of Clinical Chemistry, Burtis and Ashwood (eds.), 4thedition 1996, W.B. Saunders Company, pages 192-199; and Zweig et al.,“ROC Curve Analysis: An Example Showing The Relationships Among SerumLipid And Apolipoprotein Concentrations In Identifying Patients WithCoronory Artery Disease,” Clin. Chem., 1992, 38(8): 1425-1428.

An ROC curve is an x-y plot of sensitivity on the y-axis, on a scale ofzero to one (i.e., 100%), against a value equal to one minus specificityon the x-axis, on a scale of zero to one (i.e., 100%). In other words,it is a plot of the true positive rate against the false positive ratefor that test, assay, or method. To construct the ROC curve for thetest, assay, or method in question, patients are assessed using aperfectly accurate or “gold standard” method that is independent of thetest, assay, or method in question to determine whether the patients aretruly positive or negative for the disease, condition, or syndrome (forexample, coronary angiography is a gold standard test for the presenceof coronary atherosclerosis). The patients are also tested using thetest, assay, or method in question, and for varying cut points, thepatients are reported as being positive or negative according to thetest, assay, or method. The sensitivity (true positive rate) and thevalue equal to one minus the specificity (which value equals the falsepositive rate) are determined for each cut point, and each pair of x-yvalues is plotted as a single point on the x-y diagram. The “curve”connecting those points is the ROC curve.

The area under the curve (“AUC”) is the indicator that allowsrepresentation of the sensitivity and specificity of a test, assay, ormethod over the entire range of cut points with just a single value. Themaximum AUC is one (a perfect test) and the minimum area is one half.The closer the AUC is to one, the better is the accuracy of the test.

By a “high degree of diagnostic accuracy” is meant a test or assay (suchas the test of the invention for determining the clinically significantpresence of PD-1 polypeptide, which thereby indicates the presence ofdiabetes) in which the AUC (area under the ROC curve for the test orassay) is at least 0.70, desirably at least 0.75, more desirably atleast 0.80, preferably at least 0.85, more preferably at least 0.90, andmost preferably at least 0.95.

By a “very high degree of diagnostic accuracy” is meant a test or assayin which the AUC (area under the ROC curve for the test or assay) is atleast 0.875, desirably at least 0.90, more desirably at least 0.925,preferably at least 0.95, more preferably at least 0.975, and mostpreferably at least 0.98.

Optionally, expression of other known biomarkers for a particular cancerare also determined as further indication of whether or not the subjectis carrying a cancer. For example, CD10, bcl-6, CD20, CD57 or CXCR5 isdetected.

The PD-1 polypeptide and the additional biomarkers are detected in anysuitable manner, but are typically detected by contacting a sample fromthe patient with an antibody which binds the PD-1 or biomarker and thendetecting the presence or absence of a reaction product. The antibodymay be monoclonal, polyclonal, chimeric, or a fragment of the foregoing,as discussed in detail above, and the step of detecting the reactionproduct may be carried out with any suitable immunoassay. The samplefrom the subject is typically a biological fluid as described above, andmay be the same sample of biological fluid used to conduct the methoddescribed above.

Expression of a PD-1 polypeptide also allows for the course of treatmentof cancer to be monitored. In this method, a biological sample isprovided from a subject undergoing treatment, e.g., surgical,chemotherapeutic or hormonal treatment, for a cancer. If desired,biological samples are obtained from the subject at various time pointsbefore, during, or after treatment. Expression of a PD-1 is thendetermined and compared to a reference, e.g. control whose cancer stateis known. The reference sample has been exposed to the treatment.Alternatively, the reference sample has not been exposed to thetreatment. Optionally, such monitoring is carried out preliminary to asecond look surgical surveillance procedures and subsequent surgicalsurveillance procedures. For example, samples may be collected fromsubjects who have received initial surgical treatment for cancer andsubsequent treatment with anti-neoplastic agents for that cancer tomonitor the progress of the treatment.

If the reference sample is from a subject that does not have cancer, asimilarity or a decrease in the amount of the PD-1 polypeptide in thetest sample and the reference sample indicates that the treatment isefficacious. However, an increase in the amount of the PD-1 polypeptidein the test sample and the reference sample indicates a less favorableclinical outcome or prognosis.

By “efficacious” is meant that the treatment leads to a decrease in theamount of a PD-1 polypeptide, or a decrease in size, prevalence, ormetastatic potential of a tumor in a subject. Assessment of cancer ismade using standard clinical protocols. Efficacy is determined inassociation with any known method for diagnosing or treating theparticular tumor. Expression of a PD-1 polypeptide also allows theidentification of patients who will be responsive to systemic, e.g.,chemotherapeutic, hormonal or radiation therapy. In this method, abiological sample is provided from a subject prior to undergoingsurgical treatment, for a cancer. Expression of a PD-1 polypeptide isthen determined and compared to a biological sample obtained from thesubject after surgical removal of the cancer. The patient will likely beresponsive to systemic treatment if the amount of the PD-1 polypeptidedecreases after surgical removal the cancer. In contrast a the patientwill likely not be responsive to systemic treatment if the amount of thepolypeptide remains constant or increase after surgical removal of thecancer.

Expression of the PD-1 polypeptide or other cancer biomarkers isdetermined at the protein or nucleic acid level using any method knownin the art. For example, Northern hybridization analysis using probeswhich specifically recognize one or more of these sequences can be usedto determine gene expression. Alternatively, expression is measuredusing reverse-transcription-based PCR assays, e.g., using primersspecific for the differentially expressed sequence of genes. Expressionis also determined at the protein level, i.e., by measuring the levelsof peptides encoded by the gene products described herein, or activitiesthereof. Such methods are well known in the art and include, e.g.,immunoassays based on antibodies to proteins encoded by the genes. Anybiological material can be used for the detection/quantification of theprotein or it's activity. Alternatively, a suitable method can beselected to determine the activity of proteins encoded by the markergenes according to the activity of each protein analyzed.

The subject is preferably a mammal. The mammal is, e.g., a human,non-human primate, mouse, rat, dog, cat, horse, or cow. Subjects aretypically human females or human males The subject has been previouslydiagnosed as carrying a cancer, and possibly has already undergonetreatment for the cancer. Alternatively, the subject has not beenpreviously diagnosis as carrying a cancer. The present invention isuseful with all patients at risk for a cancer. Although each type ofcancer has its own set of risk factors, the risk of developing cancerincreases as with aged, gender, race and personal and family medicalhistory. Other risk factors are largely related to lifestyle choices,while certain infections, occupational exposures and some environmentalfactors can also be related to developing cancer.

Diagnosis of cancer is typically made through the identification of amass on an examination, though it may also be through other means suchas a radiological diagnosis, or ultrasound. Treatment is typicallythrough cytoreductive surgery, followed by treatment with antineoplasticagents such as docetaxel, vinorelbine gemcitabine, capecitabine or acombinations of cyclophosphamide, methotrexate, and fluorouracil;cyclophosphamide, doxorubicin, and fluorouracil; doxorubicin andcyclophosphamide; doxorubicin and cyclophosphamide with paclitaxel;doxorubicin followed by CMF; or Cyclophosphamide, epirubicin andfluorouracil. In addition, many patients will require radiation therapy.

Immunoassays carried out in accordance with the present invention may behomogeneous assays or heterogeneous assays. In a homogeneous assay theimmunological reaction usually involves the specific antibody (e.g.,PD-1 polypeptide), a labeled analyte, and the sample of interest. Thesignal arising from the label is modified, directly or indirectly, uponthe binding of the antibody to the labeled analyte. Both theimmunological reaction and detection of the extent thereof are carriedout in a homogeneous solution. Immunochemical labels which may beemployed include free radicals, radioisotopes, fluorescent dyes,enzymes, bacteriophages, or coenzymes.

In a heterogeneous assay approach, the reagents are usually the sample,the antibody, and means for producing a detectable signal. Samples asdescribed above may be used. The antibody is generally immobilized on asupport, such as a bead, plate or slide, and contacted with the specimensuspected of containing the antigen in a liquid phase. The support isthen separated from the liquid phase and either the support phase or theliquid phase is examined for a detectable signal employing means forproducing such signal. The signal is related to the presence of theanalyte in the sample. Means for producing a detectable signal includethe use of radioactive labels, fluorescent labels, or enzyme labels. Forexample, if the antigen to be detected contains a second binding site,an antibody which binds to that site can be conjugated to a detectablegroup and added to the liquid phase reaction solution before theseparation step. The presence of the detectable group on the solidsupport indicates the presence of the antigen in the test sample.Examples of suitable immunoassays are radioimmunoassays,immunofluorescence methods, or enzyme-linked immunoassays.

Those skilled in the art will be familiar with numerous specificimmunoassay formats and variations thereof, which may be useful forcarrying out the method disclosed herein. See generally E. Maggio,Enzyme-Immunoassay, (1980) (CRC Press, Inc., Boca Raton, Fla.); see alsoU.S. Pat. No. 4,727,022 to Skold et al. titled “Methods for ModulatingLigand-Receptor Interactions and their Application,” U.S. Pat. No.4,659,678 to Forrest et al. titled “Immunoassay of Antigens,” U.S. Pat.No. 4,376,110 to David et al., titled “Immunometric Assays UsingMonoclonal Antibodies,” U.S. Pat. No. 4,275,149 to Litman et al., titled“Macromolecular Environment Control in Specific Receptor Assays,” U.S.Pat. No. 4,233,402 to Maggio et al., titled “Reagents and MethodEmploying Channeling,” and U.S. Pat. No. 4,230,767 to Boguslaski et al.,titled “Heterogeneous Specific Binding Assay Employing a Coenzyme asLabel.” Antibodies are conjugated to a solid support suitable for adiagnostic assay (e.g., beads, plates, slides or wells formed frommaterials such as latex or polystyrene) in accordance with knowntechniques, such as precipitation. Antibodies as described herein maylikewise be conjugated to detectable groups such as radiolabels (e.g.,35 S, 125 I, 131 I), enzyme labels (e.g., horseradish peroxidase,alkaline phosphatase), and fluorescent labels (e.g., fluorescein) inaccordance with known techniques.

Diagnostic kits for carrying out the methods described herein areproduced in a number of ways. In one embodiment, the diagnostic kitcomprises (a) an antibody (e.g., PD-1 polypeptide) conjugated to a solidsupport and (b) a second antibody of the invention conjugated to adetectable group. The reagents may also include ancillary agents such asbuffering agents and protein stabilizing agents, e.g., polysaccharidesand the like. The diagnostic kit may further include, where necessary,other members of the signal-producing system of which system thedetectable group is a member (e.g., enzyme substrates), agents forreducing background interference in a test, control reagents, apparatusfor conducting a test, and the like. Alternatively, a test kit contains(a) an antibody, and (b) a specific binding partner for the antibodyconjugated to a detectable group. Ancillary agents as described abovemay likewise be included. The test kit may be packaged in any suitablemanner, typically with all elements in a single container along with asheet of printed instructions for carrying out the test.

This invention is based in part on the experiments described in thefollowing examples. These examples are provided to illustrate theinvention and should not be construed as limiting.

Example 1: Inhibition of the PD-1 Pathway in Chronically-Infected MiceUsing Anti-PD-L1 Antibodies

Mice infected with various strains of the lymphocytic choriomeningitisvirus (LCMV) were used to study the effect of chronic viral infection onCD8⁺ T cell function. The LCMV Armstrong strain causes an acuteinfection that is cleared within 8 days, leaving behind a long-livedpopulation of highly functional, resting memory CD8⁺ T cells. The LCMVC1-13 strain, in contrast, establishes a persistent infection in thehost, characterized by a viremia that lasts up to 3 months. The virusremains in some tissues indefinitely and antigen specific CD8⁺ T cellsbecome functionally impaired. D^(b)NP396-404 CD8⁺ T cells are physicallydeleted, while D^(b)GP33-41 and D^(b)GP276-286 CD8⁺ T cells persist butlose the ability to proliferate or secrete anti-viral cytokines, such asIFN-γ and TNF-α.

C57BL/6 mice were purchased from the National Cancer Institute(Frederick, Md.). Mice were infected i.v. with 2×10⁶ pfu of LCMV-Cl-13.CD4 depletions were performed by injecting 500 μg of GK1.5 in PBS theday of infection and the day following the infection. LCMV immune miceare generated by infecting mice i.p. with 2×10⁵ pfu LCMV Armstrong.

Gene array analysis was performed on FACS-purified naïve D^(b)GP33-41specific P14 transgenic CD8⁺ T cells, D^(b)GP33-41 specific memory CD8⁺T cells derived from LCMV Armstrong immune mice, and D^(b)GP33-41specific or D^(b)GP276-286 specific CD8⁺ T cells derived from CD4⁺depleted LCMV C1-13 infected mice. RNA isolation and gene array analysiswere performed as described in Kaech et al., (Cell 111:837-51, 2002).PD-1 mRNA was highly expressed in exhausted CD8⁺ T cells relative tomemory CD8⁺ T cells (FIG. 1A). Furthermore, PD-1 was expressed on thesurface of CD8⁺ T cells in LCMV C1-13 infected mice, but was not presenton the surface of CD8⁺ T cells after clearance of LCMV Armstrong (FIG.1B). Chronically infected mice also expressed higher levels of one ofthe ligands of PD-1, PD-L1, on most lymphocytes and APC compared touninfected mice. Thus, viral antigen persistence and CD8⁺ T cellexhaustion are concomitant with an induction in PD-1 expression.

To test the hypothesis that blocking the PD-1/PD-L1 pathway may restoreT cell function and enhance viral control during chronic LCMV infection,the PD-1/PD-L1 co-inhibitory pathway was disrupted during chronic LCMVinfection using αPD-L1 blocking antibodies. A blocking monoclonalantibody against PD-L1 was administered i.p. every third day to miceinfected with LCMV C1-13 (200 μg of rat anti-mouse PD-L1 IgG2bmonoclonal antibodies (clone 10F.5C5 or 10F.9G2)) from day 23 to day 37post-infection. At day 37, there was approximately 2.5 fold moreD^(b)NP396-404 specific CD8⁺ T cells and 3 fold more D^(b)GP33-41specific CD8⁺ T cells in treated mice relative to the untreated controls(FIG. 2A). The induction in proliferation was specific to CD8⁺ T cellssince the number of CD4+ T cells in the spleen were approximately thesame in both treated mice and untreated mice (˜6×10⁴ IA^(b)GP61-80 ofCD4+ T cells per spleen).

In addition to an increase in CD8+ T cell proliferation, the inhibitionof PD-1 signaling also resulted in an increased production of anti-viralcytokines in virus-specific CD8⁺ T cells. The production of IFN-γ andTNF-α by CD8+ T cells to eight different CTL epitopes was determined.The combined response was 2.3 fold higher in treated mice as compared tountreated mice (FIGS. 2B and 2C). A 2-fold increase in the frequency ofTNF-α producing cells was also observed following treatment (FIG. 2D).Viral clearance was also accelerated as the virus was cleared from theserum, spleen, and liver of treated mice. Reduced viral titers wereobserved in the lung and kidney (˜10 fold) by day 37 post-infection (14days following initiation of treatment) in treated mice. Untreated mice,however, displayed significant levels of virus in all these tissues(FIG. 2E). Viral titers in serum and tissue homogenates were determinedusing Vero cells, as described in Ahmed et al. (J. Virol. 51:34-41,1984). The results showing that a PD-1 inhibitor increases CD8+ T cellproliferation and viral clearance therefore indicate that the inhibitionof PD-1 signaling restores CD8+ T cell function. Furthermore, inhibitionof PD-1 signaling also enhanced B cell responses as the number of LCMVspecific antibody secreting cells in the spleen was also increased(>10-fold) following treatment.

CD4⁺ T cells play a key role in the generation and maintenance of CD8+ Tcell responses. In this regard, CD8+ T cells primed in the absence ofCD4+ T cell (so-called “helpless” CD8+ T cells) are incapable ofmounting normal immune responses. Furthermore, chronic LCMV infection ismore severe in the absence of CD4+ T cells. Accordingly, helpless Tcells generated during LCMV-Cl-13 infection display an even moreprofound functional impairment than T cells generated in the presence ofCD4+ T cells. D^(b)NP396-404 specific CD8⁺ T cells are deleted toundetectable levels, and D^(b)GP33-41 and D^(b)GP276-286 CD8⁺ T cellscompletely lose the ability to secrete IFN-γ and TNF-α.

CD4⁺ T cells were depleted at the time of LCMV-Cl-13 infection and micewere treated with anti-PD-L antibodies treatment from day 46 to day 60post-infection. LCMV-specific CD4⁺ T cells were not detectable byintracellular IFN-γ staining before or after treatment. Followingtreatment, treated mice had approximately 7 fold more D^(b)GP276-286CD8⁺ T cells and 4 fold more D^(b)GP33-41 CD8⁺ T cells in their spleenthan untreated control mice (FIG. 3A). The number of virus-specific CD8⁺T cells in the spleen was also increased (FIG. 3B). This increase invirus-specific CD8⁺ T cells in treated mice was attributed to anincrease in proliferation, as detected by BrdU incorporation. 43% ofD^(b)GP276-286 CD8⁺ T cells incorporated intermediate levels of BrdU and2% incorporated high levels of BrdU in untreated mice, while 50%D^(b)GP276-286 CD8⁺ T cells incorporated intermediate levels of BrdU and37% incorporated high levels of BrdU in treated mice. BrdU analysis wasperformed by introducing 1 mg/ml BrdU in the drinking water duringtreatment and staining was performed according to the manufacturer'sprotocol (BD Biosciences, San Diego, Calif.). Moreover, treated micecontained a higher percentage of CD8⁺ T cells expressing the cellcycle-associated protein Ki67 (60% versus 19% in untreated mice, FIG.3C). Response to treatment in CD8⁺ T cells in the PBMC was restricted tomice having high levels of CD8⁺ T cell expansion.

PD-1 inhibition also increased anti-viral cytokine production inhelpless, exhausted virus-specific CD8⁺ T cells. Following treatment,the number of D^(b)GP33-41 and D^(b)GP276-286 CD8⁺ T cells that produceIFN-γ was markedly increased (FIG. 4A), though higher numbers ofD^(b)NP396-404, K^(b)NP205-212, D^(b)NP166-175, and D^(b)GP92-101specific CD8+ T cells were also detected in treated mice (FIG. 4A). 50%of D^(b)GP276-286 specific CD8⁺ T cells from treated mice can produceIFN-γ compared to the 20% of D^(b)GP276-286 specific CD8⁺ T cells incontrol untreated mice. (FIG. 4B). Levels of IFN-γ and TNF-α produced byD^(b)GP276-286 specific CD8⁺ T cells from treated mice, however, werelower than fully functional D^(b)GP276-286 specific memory cells. (FIG.4C).

PD-1 inhibition also increased the lytic activity of helpless, exhaustedvirus-specific CD8⁺ T cells. Ex vivo lytic activity of virus-specificCD8⁺ T cells was detected following treatment, using a ⁵¹Cr releaseassay (Wherry et al., 2003. J. Virol. 77:4911-27). Viral titers werereduced by approximately 3 fold in the spleen, 4 fold in the liver, 2fold in the lung, and 2 fold in serum after 2 weeks of treatmentrelative to untreated mice. (FIG. 4E).

These results therefore demonstrate that blocking the PD-1 pathwaybreaks CTL peripheral tolerance to a chronic viral infection, and thatexhausted CD8⁺ T cells deprived of CD4⁺ T cell help are not irreversiblyinactivated.

Example 2: Administration of Anti-Viral Vaccine and PD-1 Inhibitor

One approach for boosting T cell responses during a persistent infectionis therapeutic vaccination. The rationale for this approach is thatendogenous antigens may not be presented in an optimal or immunogenicmanner during chronic viral infection and that providing antigen in theform of a vaccine may provide a more effective stimulus forvirus-specific T and B cells. Using the chronic LCMV model, mice wereadministered a recombinant vaccinia virus expressing the LCMV GP33epitope as a therapeutic vaccine (VVGP33), which resulted in a modestenhancement of CD8+ T cell responses in some chronically infected miceFour out of the nine chronically infected mice that received thetherapeutic vaccine showed a positive response while none of the controlmice had a significant increase in the immune response against GP33.When this therapeutic vaccination was combined with a PD-L1 inhibitor,LCMV specific T cell responses were boosted to a greater level thancompared to either treatment alone and the effect of combined treatmentwas more than additive.

Example 3: Inhibition of the PD-1 Pathway in Chronically-Infected MiceUsing PD-1 RNAi

RNA interference (RNAi) is capable of silencing gene expression inmammalian cells. Long double stranded RNAS (dsRNAs) are introduced intocells and are next processed into smaller, silencing RNAs (siRNAs) thattarget specific mRNA molecules or a small group of mRNAs. Thistechnology is particularly useful in situations where antibodies are notfunctional. For example, RNAi may be employed in a situation in whichunique splice variants produce soluble forms of PD-1 and CTLA-4.

PD-1 silencer RNAs are inserted into a commercially available siRNAexpression vector, such as pSilencer™ expression vectors or adenoviralvectors (Ambion, Austin, Tex.). These vectors are then contacted withtarget exhausted T cells in vivo or ex vivo (See, Example 4).

Example 4: Ex Vivo Rejuvenation of Exhausted T Cells

Virus-specific exhausted CD8⁺ T cells are isolated from LCMV-Cl-13chronically infected mice using magnetic beads or densitycentrifugation. Transfected CD8⁺ T cells are contacted with a monoclonalantibody that targets PD-L1, PD-L2 or PD-1. As described in Example 1,inhibition of the PD-1 pathway results in the rejuvenation of the CD8⁺ Tcells. Accordingly, there is an increase in CD8+ T cell proliferationand cytokine production, for example. These rejuvenated CD8⁺ T cells arereintroduced into the infected mice and viral load is measured asdescribed in Example 1.

Example 5: In Vitro Screening of Novel CD8⁺ T Cell Rejuvenator Compounds

Compounds that modulate the PD-1 pathway can be identified in in vivoand ex vivo screening assays based on their ability to reverse CD8⁺ Tcell exhaustion resulting from chronic viral infection.

Exhausted CD8⁺ T cells are derived from mice chronically infected withLCMV-Cl-13 and next contacted with a test compound. The amount ofanti-viral cytokines (e.g., IFN-γ or TNF-α) released from the contactedT cell is measured, for example, by ELISA or other quantitative method,and compared to the amount, if any, of the anti-viral cytokine releasedfrom the exhausted T cell not contacted with the test compound. Anincrease in the amount of anti-viral cytokine released by treated cellsrelative to such amount in untreated cells identifies the compound as aPD-1 inhibitor, useful to modulate T cell activity.

Example 6: In Vivo Screening of Novel CD8⁺ T Cell Rejuvenator Compounds

Exhausted CD8⁺ T cells are derived from mice chronically infected withLCMV-Cl-13. A test compound is administered intravenously to theinfected mice. The amount of anti-viral cytokines (e.g., IFN-γ or TNF-α)that is released into the serum of treated and untreated mice ismeasured, for example, by ELISA or other quantitative method, andcompared. An increase in the amount of anti-viral cytokine found in theserum in treated mice relative to such amount in untreated miceidentifies the test compound as a PD-1 inhibitor. Alternatively, theviral titer (e.g., serum viral titer) can be determined prior andsubsequent to treatment of the test compound.

Example 7: Chimpanzees as a Model for Immunotherapy of Persistent HCVInfection

Chimpanzees provide a model of HCV persistence in humans. Defects in Tcell immunity leading to life-long virus persistence both include adeficit in HCV-specific CD4+T helper cells and impaired or altered CD8+Teffector cell activity. Persistently infected chimpanzees are treatedwith antibodies against CTLA-4, PD-1, or a combination of the two. Theefficacy of blockade of the inhibitory pathways, combined withvaccination using recombinant structural and non-structural HCVproteins, and whether such strategies can enhance the frequency andlongevity of virus-specific memory T cells are determined. The defect inT cell immunity is exclusively HCV-specific in persistently infectedhumans and chimpanzees. The blood and liver of infected chimpanzees areexamined for expression of CTLA-4, PD-1, BTLA and their ligands and forthe presence of Treg cells. Antiviral activity may then be restored bydelivering to chimpanzees humanized monoclonal antibodies that blocksignaling through these molecules.

Persistently infected chimpanzees are treated with humanized αCTLA-4antibodies (MDX-010, Medarex) or αPD-1 antibodies. The initial dose ofMDX-010 is 0.3 mg/kg followed 2 weeks later by 1.0 mg/kg and then 3, 10,30 mg/kg at three week intervals. After treatment with antibodies toco-inhibitory molecules, the humoral and cellular immune responses aswell as the HCV RNA load will be determined. Samples are collected atweeks 1, 2, 3, 5, and 8, and then at monthly intervals. Samplesinclude: 1) serum for analysis of transaminases, autoantibodies,neutralizing antibodies to HCV, and cytokine responses, 2) plasma forviral load and genome evolution, 3) PBMC for in vitro measures ofimmunity, costimulatory/inhibitory receptor expression and function, 4)fresh (unfixed) liver for isolation of intrahepatic lymphocytes and RNA,and 5) fixed (formalin/paraffin embedded) liver for histology andimmunohistochemical analysis. Regional lymph nodes are also collected at2 or 3 time points to assess expression of co-inhibitory molecules andsplice variants by immunohistochemistry and molecular techniques. Assaysto evaluate the efficacy and safety of these therapies safety will beperformed as described herein.

To determine if vaccination with HCV antigens potentiates thetherapeutic effect of antibodies to PD-1, chimpanzees are treated asfollows: 1) intramuscular immunization with recombinant envelopeglycoproteins E1 and E2 (in MF59 adjuvant) and other proteins (core plusNS 3, 4, and 5 formulated with ISCOMS) at weeks 0, 4, and 24; 2)intramuscular immunization with the vaccine used in 1) butco-administered with αCTLA-4 antibodies (30 mg of each/Kg body weight,intravenously at weeks 0, 4, and 24 when vaccine is given); 3) identicalto 2) except that αPD-1 (or BTLA) antibodies are substituted for theCTLA-4 antibodies; 4) identical to Groups 2 and 3 except that acombination of CTLA-4 and PD-1 (or BTLA) antibodies are used in additionto the vaccine. HCV-specific T and B cell responses are monitored atmonthly intervals after immunization for a period of 1 year.

Markers examined on HCV-tetramer+ and total T cells in this analysisinclude markers of differentiation (e.g. CD45RA/RO, CD62L, CCR7, andCD27), activation (e.g. CD25, CD69, CD38, and HLA-DR),survival/proliferation (e.g. bcl-2 and Ki67), cytotoxic potential (e.g.granzymes and perforin), and cytokine receptors (CD122 and CD127). Aninteresting correlation exists between pre-therapy levels of thechemokine IP-10 and response to PEG IFN-γ/ribavirin. IP-10 levels aremeasured to investigate a potential correlation between negativeregulatory pathways or HCV-specific T cell responses and IP-10 levels.Expression of inhibitory receptors and ligands on PBMC are performed byflow cytometry.

Example 8: PD-1 Immunostaining in Reactive Lymphoid Tissue

Materials

Case material was obtained from the Brigham & Women's Hospital, Boston,Mass., in accordance with institutional policies. All diagnoses werebased on the histologic and immunophenotypic features described in theWorld Health Organization Lymphoma Classification system (Jaffe E S, etal. 2001) and in all cases diagnostic material was reviewed by ahematopathologist.

Immunostaining

Immunostaining for PD-1 was performed on formalin-fixed paraffinembedded tissue sections following microwave antigen retrieval in 10 mMcitrate buffer, pH 6.0 with a previously described anti-human PD-1monoclonal antibody (2H7; 5), using a standard indirect avidin-biotinhorseradish peroxidase method and diaminobenzidine color development, aspreviously described (Jones D, et al. 1999; Dorfman D M, et al. 2003).Cases were regarded as immunoreactive for PD-1 if at least 25% ofneoplastic cells exhibited positive staining. PD-1 staining was comparedwith that of mouse IgG isotype control antibody diluted to identicalprotein concentration for all cases studied, to confirm stainingspecificity.

Monoclonal antibody 2H7 for PD-1 was used to stain formalin-fixed,paraffin-embedded specimens of reactive lymphoid tissue, thymus, and arange of cases of B cell and T cell lymphoproliferative disorders. Inspecimens of tonsil exhibiting reactive changes, including follicularhyperplasia, a subset of predominantly small lymphocytes in the germinalcenters exhibited cytoplasmic staining for PD-1, with infrequentPD-1-positive cells seen in the interfollicular T cell zones. The PD-1staining pattern in germinal centers was virtually identical to thatseen with an antibody to CD3, a pan-T cell marker, whereas an antibodyto CD20, a pan-B cell marker, stained the vast majority of germinalcenter B cells. Similar results were seen in histologic sections ofreactive lymph node and spleen. No PD-1 staining was observed in adultthymus.

Example 9: PD-1 Immunostaining in Paraffin Embedded Tissue Sections of BCell and T Cell Lymphoproliferative Disorders

A range of B cell and T cell lymphoproliferative disorders for PD-1expression were studied; the results are summarized in Table 1.Forty-two cases of B cell lymphoproliferative disorders were examinedfor PD-1 expression, including representative cases of precursor Blymphoblastic leukemia/lymphoblastic lymphoma, as well as a range oflymphoproliferative disorders of mature B cells, including a number of Bcell non-Hodgkin lymphomas of follicular origin, including 6 cases offollicular lymphoma and 7 cases of Burkitt lymphoma. None of the B celllymphoproliferative disorders showed staining for PD-1. In some cases,non-neoplastic reactive lymphoid tissue was present, and showed a PD-1staining pattern as seen in tonsil and other reactive lymphoid tissuenoted above.

Similarly, in 25 cases of Hodgkin lymphoma, including 11 cases ofclassical Hodgkin lymphoma and 14 case of lymphocyte predominant Hodgkinlymphoma, the neoplastic cells did not exhibit staining for PD-1.Interestingly, in all 14 cases of lymphocyte predominant Hodgkinlymphoma, the T cells surrounding neoplastic CD20-positive L&H cellswere immunoreactive for PD-1, similar to the staining pattern noted forCD57+ T cells in lymphocyte predominant Hodgkin lymphoma. ThesePD-1-positive cells were a subset of the total CD3+ T cell populationpresent.

A range of T cell lymphoproliferative disorders were studied forexpression of PD-1; the results are summarized in Table 1. Cases ofprecursor T cell lymphoblastic leukemia/lymphoblastic lymphoma, aneoplasm of immature T cells of immature T cells, were negative forPD-1, as were neoplasms of peripheral, post-thymic T cells, includingcases of T cell prolymphocytic leukemia, peripheral T cell lymphoma,unspecified, anaplastic large cell lymphoma, and adult T cellleukemia/lymphoma. In contrast, all 19 cases of angioimmunoblasticlymphoma contained foci of PD-1-positive cells that were alsoimmunoreactive for pan-T cell markers such as CD3. PD-1-positive cellswere consistently found at foci of expanded CD21+ follicular dendriticcells (FDC) networks, a characteristic feature of angioimmunoblasticlymphoma.

TABLE 1 PD-1 immunostaining in lymphoproliferative disorders. PD-1immunostaining B cell LPDs 0/42*  B-LL/LL 0/3   CLL 0/4   MCL 0/4   FL0/6   MZL 0/3   HCL 0/3   DLBCL 0/6   BL 0/7   LPL 0/3   MM 0/3  Hodgkin lymphoma 0/25  Classical 0/11  Nodular lymphocyte 0/14**predominant T cell LPDs 18/55   T-LL/LL 0/5   T-PLL 0/3   AIL 19/19  PTCL, unspecified 0/14  ALCL 0/12  ATLL 0/3   Abbreviations:B-LL/LL—precursor B cell lymphoblastic lymphoma/lymphoblastic leukemia;CLL—chronic lymphocytic leukemia; MCL—mantle cell lymphoma;FL—follicular lymphoma; MZL—marginal zone lymphoma; HCL—hairy cellleukemia; DLBCL—diffuse large B cell lymphoma; BL—Burkitt lymphoma;LPL—lymphoplasmacytic lymphoma; MM—multiple myeloma; T-LL/L—precursor Tlymphoblastic leukemia/lymphoblastic lymphoma; T-PLL—T cellprolymphocytic leukemia; AIL—angioimmunoblastic lymphoma;PTCL—peripheral T cell lymphoma, unspecified; ALCL—anaplastic large celllymphoma; ATLL—adult T cell leukemia/lymphoma. *number of immunoreactivecases/total number of cases **PD-1-positive cells form rosettes aroundneoplastic L&H cells in 14/14 cases

Example 10: General Methods for Studying PD-1 Expression on HIV-SpecificHuman CD8 T Cells

The following methods were used to perform the experiments detailed innExamples 11-14.

Study Subjects

Study participants with chronic clade C HIV-1 infection were recruitedfrom outpatient clinics at McCord Hospital, Durban, South Africa, andSt. Mary's Hospital, Mariannhill, South Africa. Peripheral blood wasobtained from 65 subjects in this cohort, all of whom wereantiretroviral therapy naïve at the time of analysis. Subjects wereselected for inclusion based on their expressed HLA alleles matching theten class I tetramers that were constructed (see below). The medianviral load of the cohort was 42,800 HIV-1 RNA copies/ml plasma (range163-750,000), and the median absolute CD4 count was 362 (range129-1179). Information regarding duration of infection was notavailable. All subjects gave written informed consent for the study,which was approved by local institutional review boards.

Construction of PD-1 and PD-L1 Antibodies

Monoclonal antibodies to human PD-L1 (29E.2A3, mouse IgG2b) and PD-1(EH12, mouse IgG1) were prepared as previously described and have beenshown to block the PD-1:PD-L1 interaction.

MHC Class I Tetramers

Ten HIV MHC Class I tetramers, synthesized as previously described(Altman J D, et al. 1996), were used for this study: A*0205 GL9 (p24,GAFDLSFFL; SEQ ID NO:), A*3002 KIY9 (Integrase, KIQNFRVYY; SEQ ID NO:2),B*0801 DI8 (p24, DIYKRWII; SEQ ID NO:3), B*0801 FL8 (Nef, FLKEKGGL; SEQID NO:4), B*4201 RM9 (Nef, RPQVPLRPM; SEQ ID NO:5), B*4201 TL9 (p24,TPQDLNTML; SEQ ID NO:6), B*4201 TL10 (Nef, TPGPGVRYPL; SEQ ID NO:7),B*4201 YL9 (RT, YPGIKVKQL; SEQ ID NO:8), B*8101 TL9 (p24, TPQDLNTML; SEQID NO:9), and Cw0304 YL9 (p24, YVDRFFKTL; SEQ ID NO: 10). The plasmidconstructs expressing A*0205, A*3002, and Cw*0304 were kindly providedby Drs. Eugene Ravkov and John Altman, NIH Tetramer Core Facility,Atlanta, Ga.

HLA Class I Tetramer Staining and Phenotypic Analysis

Freshly isolated peripheral blood mononuclear cells (PBMC, 0.5 million)were stained with tetramer for 20 minutes at 37° C. The cells were thenwashed once with phosphate buffered saline (PBS), pelleted, and staineddirectly with fluorescein isothiosyanate (FITC)-conjugated anti-CD8(Becton Dickinson), phycoerythrin-conjugated anti-PD-1 (clone EH12), andViaProbe (Becton Dickinson). Cells were incubated for 20 minutes at roomtemperature, washed once in PBS, and resuspended in 200 μl PBS with 1%paraformaldehyde and acquired on a fluorescence-activated cell sorter(FACSCalibur, Becton Dickinson). A minimum of 100,000 events wereacquired on the FACSCalibur.

CFSE Proliferation Assays

One million freshly isolated PBMC were washed twice in PBS, pelleted,and resuspended in 1 ml of 0.5 μM carboxy-fluorescein diacetate,succinimidyl ester (CFSE, Molecular Probes) for 7 minutes at 37° C. Thecells were washed twice in PBS, resuspended in 1 ml R10 medium (RPMI1640 supplemented with glutiathione, penicillin, streptomycin, and 10%fetal calf serum [FCS]), and plated into one well of a 24-well plate.Initial studies revealed that a final concentration of 0.2 μg/ml peptideyielded optimal proliferative responses, therefore this was the finalpeptide concentration in the well used for each assay. Negative controlwells consisted of PBMC in medium alone, or PBMC in medium with purifiedanti-PD-L1 (10 μg/ml), and positive control wells were stimulated with10 μg/ml of phytohemagluttinin (PHA). Following 6-day incubation in a37° C. incubator, the cells were washed with 2 ml PBS and stained withPE-conjugated MHC Class I tetramers, ViaProbe (Becton Dickinson), andanti-CD8-APC antibodies. Cells were acquired on a FACSCalibur andanalyzed by CellQuest® software (Becton Dickinson). Cells were gated onViaProbe⁻ CD8⁺ lymphocytes. The fold increase in tetramer⁺ cells wascalculated by dividing the percentage of CD8⁺ tetramer⁺ cells in thepresence of peptide by the percentage of CD8⁺ tetramer⁺ cells in theabsence of peptide stimulation.

Statistical Analysis

Spearman correlation, Mann-Whitney test, and paired t-test analyses wereperformed using GraphPad Prism Version 4.0a. All tests were 2-tailed andp values of p<0.05 were considered significant.

Example 11: PD-1 Expression on HIV-Specific CD8 T Cells

A panel of 10 MHC Class I tetramers specific for dominant HIV-1 clade Cvirus CD8 T cell epitopes was synthesized, based on prevalent HLAalleles and frequently targeted epitopes in Gag, Nef, Integrase, and RT(Kiepiela P, et al. 2004), allowing direct visualization of surface PD-1expression on these cells. High resolution HLA typing was performed onthe entire cohort, and a subset of 65 antiretroviral therapy naïvepersons was selected for study based on expression of relevant HLAalleles. A total of 120 individual epitopes were examined, andrepresentative ex vivo staining of PD-1 on HIV tetramer⁺ cells is shownin FIG. 5A. PD-1 expression was readily apparent on these tetramer⁺cells, and was significantly higher than in the total CD8 T cellpopulation from the same individuals (p<0.0001); in turn, PD-1expression on both tetramer⁺ CD8 T cells and the total CD8 T cellpopulation was significantly higher than in HIV-seronegative controls(FIG. 5B). For eight of the ten tetramers tested at least one person wasidentified in whom the level of expression on antigen-specific CD8 cellswas 100% (FIG. 5C). PBMC from 3 to 25 individuals were stained for eachHIV tetramer response, with median PD-1 expression levels ranging from68% to 94% of tetramer⁺ cells (FIG. 5C). These findings were furtherconfirmed by analysis of the mean fluorescence intensity (MFI) of PD-1on both tetramer⁺ cells and the total CD8 T cell population (FIG. 5B,C).

It was next determined whether there was evidence for epitope-specificdifferences in terms of PD-1 expression levels in persons with multipledetectable responses. Of the 65 persons examined, 16 individuals hadbetween 3 and 5 tetramer positive responses each. PD-1 expression wasnearly identical and approaching 100% for each response analyzed forthree of the sixteen subjects; however, the other 13 individualsdisplayed different patterns of PD-1 expression depending on the epitope(FIG. 5D). These data indicate that PD-1 expression may bedifferentially expressed on contemporaneous epitope-specific CD8 T cellsfrom a single person, perhaps consistent with recent data indicatingepitope-specific differences in antiviral efficacy (Tsomides T J, et al.1994; Yang O, et al. 1996; Loffredo J T, et al. 2005).

Example 12: The Relationship Between PD-1 Expression and HIV DiseaseProgression

The relationship was determined between PD-1 expression on HIV-specificCD8 T cells and plasma viral load and CD4 counts, both of which arepredictors of HIV disease progression. Consistent with previous studies,the relationship between the number of tetramer positive cells and viralload or CD4 count failed to show any significant correlation (FIG. 6A,B). In contrast, there were significant positive correlations with viralload and both the percentage and MFI of PD-1 expression on HIV tetramerpositive cells (p=0.0013 and p<0.0001, respectively; FIG. 6A). Therewere also inverse correlations between CD4 count and both the percentageand MFI of PD-1 on HIV tetramer positive cells (p=0.0046 and p=0.0150,respectively; FIG. 6B). Since the tetramers tested likely represent onlya fraction of the HIV-specific CD8 T cell population in these subjects,the relationship between PD-1 expression on all CD8 cells and theseparameters was also examined. There were significant positivecorrelations between viral load and both the percentage and MFI of PD-1expression on the total CD8 T cell population (p=0.0021 and p<0.0001,respectively; FIG. 6C), and inverse correlations were also observedbetween CD4 count and both the percentage and MFI of PD-1 expression onthe total CD8 T cell population (p=0.0049 and p=0.0006, respectively;FIG. 6D). In this same group, PD-1 expression on CMV-specific CD8 Tcells was tested in 5 subjects, and significantly less PD-1 wasexpressed on these cells compared to HIV-specific CD8 T cells (median23% CMV tetramer⁺ PD-1⁺, p=0.0036, data not shown), and was notdifferent than bulk CD8 T cells in these same individuals, indicatingthat high PD-1 expression is not a uniform feature of all virus-specificCD8 T cells. These data suggest increasing amounts of antigen in chronicHIV infection result in increased expression of PD-1 on CD8 T cells, andare consistent with murine data in chronic LCMV infection, in which PD-1expression is associated with functional exhaustion of CD8 T cells(Barber D L, et al. 2005). Moreover, they provide the first clearassociation, in a large study including analysis of multiple epitopes,between HIV-specific CD8 T cells and either viral load or CD4 count.

Example 13: The Relationship Between PD-1 Expression and CD8 T CellMemory Status and Function

PD-1 expression was next analyzed in the context of a number ofadditional phenotypic markers associated with CD8 T cell memory statusand function, including CD27, CD28, CD45RA, CD57, CD62L, CD127, CCR7,perforin, granzyme B, and Ki67 (FIG. 7). Representative stainings forthese markers on B*4201 TL9 tetramer⁺ cells from one individual areshown in FIG. 7A, and aggregate data for 13 subjects are shown in FIG.7B. These studies were limited to those tetramer responses that weregreater than 95% PD-1 positive, as multiparameter flow cytometry ofgreater than 4 colors was not available in KwaZulu Natal. The HIVtetramer⁺ PD-1⁺ cells express high levels of CD27 and granzyme B, verylow levels of CD28, CCR7, and intracellular Ki67, low levels of CD45RAand perforin, and intermediate levels of CD57 and CD62L (FIG. 7B). Thesedata indicate that HIV-specific PD-1⁺ T cells display aneffector/effector memory phenotype, and are consistent with previousreports of skewed maturation of HIV-specific CD8 T cells (Champagne P,et al. 2001; Appay V, et al. 2002; Hess C, et al. 2004). In addition,virus sequencing was performed to determine whether these cells weredriving immune escape (Brown J A, et al. 2003). Of 45 of thesetetramer-positive responses evaluated, the viral epitopes in only 5 weredifferent from the South African clade C consensus sequence (data notshown), indicating these cells exert little selection pressure in vivo.

Previous experiments in mice using the LCMV model showed that in vivoblockade of PD-1/PD-L1 interaction by infusion of anti-PD-L1 blockingantibody results in enhanced functionality of LCMV-specific CD8 T cellsas measured by cytokine production, killing capacity, proliferativecapacity, and, most strikingly, reduction in viral load (Barber D L, etal. 2005).

Example 14: Effect of Blockading the PD-1/PD-L1 Pathway on Proliferationof HIV-Specific CD8 T Cells

Because HIV-specific CD8 T cells also exhibit impaired proliferativecapacity (Migueles S A, et al. 2002; Lichterfeld M, et al. 2004), it wasdetermined whether blockade of the PD-1/PD-L1 could enhance thisfunction in vitro. Representative data from a B*4201-positive individualare shown in FIG. 8A. Incubation of freshly isolated CFSE-labeled PBMCwith medium alone, or medium with anti-PD-L1 antibody, resulted inmaintenance of a population of B*4201-TL9-specific CD8 T cells (1.2% ofCD8 T cells) that remained CFSE^(hi) after six days in culture.Simulation of CFSE-labeled PBMC for 6 days with TL9 peptide aloneresulted in a 4.8-fold expansion of CFSE^(lo) B*4201 TL9 tetramer⁺cells, whereas stimulation of CFSE-labeled PBMC with TL9 peptide in thepresence of anti-PD-L1 blocking antibody further enhanced proliferationof TL9-specific cells, resulting in a 10.3-fold increase in tetramer⁺cells. CFSE proliferation assays were performed on 28 samples in thepresence and absence of purified anti-human PD-L1 blocking antibody. Asignificant increase in the proliferation of HIV-specific CD8⁺ T cellswas observed in the presence of peptide plus anti-PD-L1 blockingantibody as compared to the amount of proliferation followingstimulation with peptide alone (FIG. 8B; p=0.0006, paired t-test). Thefold increase of tetramer⁺ cells in the presence of anti-PD-L1 blockingantibody varied by individual and by epitope within a given individual(FIG. 8C), again suggesting epitope-specific differences in the degreeof functional exhaustion of these responses.

OTHER EMBODIMENTS

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

The invention claimed is:
 1. A method of increasing cytotoxic activityof anergic T cells in a subject having a lymphoproliferative cancerand/or decreasing a symptom of the lymphoproliferative cancer in thesubject, comprising administering to the subject having thelymphoproliferative cancer an effective amount of an agent that reducesthe activity of a Programmed Cell Death (PD)-1 polypeptide, wherein theagent is an anti-PD-1 antibody, thereby increasing the cytotoxicactivity of anergic T cells and/or decreasing a symptom of thelymphoproliferative cancer in the subject, wherein thelymphoproliferative cancer is an angioimmunoblastic lymphoma.
 2. Themethod of claim 1, wherein increasing the cytotoxic activity of anergicT cells comprises increasing cytokine production or T cellproliferation.
 3. The method of claim 2, wherein the cytokine isinterferon γ, tumor necrosis factor α or interleukin-2.
 4. The method ofclaim 1, further comprising measuring cytotoxic T cell activity in abiological sample from the subject.
 5. The method of claim 4, whereinmeasuring cytotoxic T cell activity comprises: a) measuring thecytotoxic activity of anergic CD8+ T cells in the biological sample fromthe subject; or b) measuring the production of a cytokine by T cells inthe biological sample from the subject.
 6. The method of claim 5,wherein the cytokine is interferon γ, tumor necrosis factor α orinterleukin-2.
 7. The method of claim 1, wherein the method furthercomprises administering to the subject an effective amount of a secondcompound that induces an immune response.
 8. The compound of claim 7,wherein the second compound is an anti-inflammatory compound, anantineoplastic compound or an analgesic.
 9. The method of claim 7,wherein the second compound is an anti-CTLA-4 antibody, an anti-BTLAantibody, or an anti-B7-H4 antibody.
 10. The method of claim 7, whereinthe second compound reduces cancer cell volume.
 11. The method of claim1, wherein the antibody is a monoclonal antibody, a humanized antibody,a deimmunized antibody, or an immunoglobulin (Ig) fusion protein. 12.The method of claim 1, wherein the subject is human.
 13. The method ofclaim 1, wherein the method decreases the symptom of thelymphoproliferative cancer in the subject.
 14. The method of claim 1,wherein the method decreases the size of the tumor or the prevalence ofcancer cells.
 15. A method of decreasing a symptom of alymphoproliferative cancer in a subject, comprising administering to thesubject having the lymphoproliferative cancer an effective amount of (a)an agent that reduces the activity of a Programmed Cell Death (PD)-1polypeptide, wherein the agent is an anti-PD-1 antibody or ananti-Programmed Cell Death Ligand (PD-L1) antibody, and (b) ananti-CTLA-4 antibody, thereby decreasing the symptom of thelymphoproliferative cancer, wherein the lymphoproliferative cancer is aHodgkin's lymphoma or angioimmunoblastic lymphoma.
 16. The method ofclaim 15, wherein the lymphoproliferative cancer is the Hodgkin'slymphoma.
 17. The method of claim 15, wherein the Hodgkin's lymphoma isa nodular lymphocyte predominant Hodgkin's lymphoma.
 18. The method ofclaim 15, wherein the subject is human.
 19. The method of claim 15,wherein the lymphoproliferative cancer is the angioimmunoblasticlymphoma, and wherein the method reduces the symptom of theangioimmunoblastic lymphoma.
 20. The method of claim 15, wherein a) theanti-PD-L1 antibody is a monoclonal antibody, a humanized antibody, adeimmunized antibody, or an immunoglobulin (Ig) fusion protein; or b)the anti-PD-1 antibody is a monoclonal antibody, a humanized antibody, adeimmunized antibody, or an Ig fusion protein.